Anti-PD-1 Antibodies

ABSTRACT

Antibodies that bind to programmed cell death protein 1 (PD-1), compositions comprising such antibodies, and methods of making and using such antibodies are disclosed.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/095,675, filed on Dec. 22, 2014, U.S. Provisional Application No.62/220,199, filed on Sep. 17, 2015, U.S. Provisional Application No.62/251,082, filed on Nov. 4, 2015, and U.S. Provisional Application No.62/261,118, filed on Nov. 30, 2015. The entire teachings of the aboveapplications are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

-   -   a) File name: 50911000004SEQUENCELISTING.txt; created Dec. 18,        2015, 73 KB in size.

BACKGROUND OF THE INVENTION

Modulation of the mammalian adaptive immune response (immunomodulation)is a useful therapeutic approach for various diseases and disorders. Oneway to achieve such immunomodulation is to intervene at one or moreimmune checkpoints, e.g., the Programmed Death-1 (PD-1) checkpoint. Thenatural function of immune checkpoints is to suppress the immuneresponse, as necessary, to prevent immune damage to normal tissue.Depending on the disease or disorder, it may be desirable to upregulateor downregulate the immune response. Tumor cells that displaynon-self-antigens can evade immune attack by secreting cytokines orligands that activate immune checkpoints. Therefore, in cancer therapy,it is generally desirable to upregulate the immune response againsttumor cells. In contrast, in treatment of autoimmune diseases, it isgenerally desirable to downregulate the immune response in certaintissues.

“Programmed Death-1” (PD-1) protein (also known as Programmed Cell DeathProtein 1 and CD279) is a type I transmembrane receptor that is part ofthe extended CD28/CTLA4 family of T cell regulators. Ligands for PD-1include PD-1 Ligand 1 (PD-L1, also known as B7-H1), and PD-1 Ligand 2(PD-L2, also known as B7-DC).

PD-1 is expressed on various cell types, including T cells, B cells, andmacrophages. Experimental data implicate the interactions of PD-1 withits ligands in downregulation of central and peripheral immuneresponses. Proliferation of T cells is inhibited in the presence ofPD-L1. Mice with a disrupted PD-1 gene exhibit an autoimmune phenotype.PD-1 deficiency in the C57BL/6 mice results in chronic progressivelupus-like glomerulonephritis and arthritis (Nishimura et al., J. Exp.Med. 101(5):891-98, 2000).

Compounds that modulate PD-1 activity have potential as therapeuticagents for the treatment of various diseases and disorders, includingcancer, inflammation, and autoimmune diseases. There is a significantunmet need for immunomodulatory compounds, e.g., antibodies, includingPD-1 agonists and PD-1 antagonists.

SUMMARY OF THE INVENTION

The present invention provides antibodies that bind to PD-1. In someembodiments, the invention provides an isolated antibody that binds toPD-1, comprising a heavy chain variable region (HCVR) selected from thegroup consisting of SEQ ID NOs: 1-26 and/or a light chain variableregion (LCVR) selected from the group consisting of SEQ ID NOs: 27-53.The invention also provides an isolated antibody that binds to PD-1 andcompetitively inhibits the binding of any of the antibodies disclosedherein to PD-1.

In some embodiments, the invention also provides an isolated antibodythat binds to PD-1, comprising a HCVR selected from the group consistingof SEQ ID NOs: 85-90 and/or a LCVR selected from the group consisting ofSEQ ID NOs: 91-96.

The invention further provides an isolated antibody that binds to PD-1,wherein the antibody binds to a sequence in PD-1 selected from the groupconsisting of SEQ ID NOs: 54-84.

The antibodies can be used as therapeutic agents. For use as therapeuticagents, the antibodies disclosed herein can be engineered, e.g.,humanized, to reduce or eliminate serum sickness or an undesired immuneresponse when administered to a human patient. Also disclosed aremethods of treating diseases and disorders in which the PD-1 signalingpathway plays a significant role (“PD-1-mediated diseases anddisorders”).

The present invention includes the surprising discovery that contactinghuman T cells with an effective amount of an anti-PD-1 antibody thatcompetitively inhibits binding of PD-L1 or PD-L2 to PD-1 expressed onthe surface of T cells, and an effective amount of an anti-PD-1 antibodythat does not competitively inhibit binding of PD-L1 or PD-L2 to PD-1expressed on the surface of the T cells increases T cell effectorfunction to a greater extent than an equivalent amount of eitheranti-PD-1 antibody alone. In some embodiments, the combination yields anadditive effect on T cell effector function. In some embodiments, thecombination yields a synergistic effect on T cell effector function.

Accordingly, the present invention provides a method for increasing Tcell effector function, comprising contacting a T cell with acombination of: (a) an effective amount of an anti-PD-1 antibody thatcompetitively inhibits binding of PD-L1 or PD-L2 to PD-1 expressed onthe surface of the T cell; and (b) an effective amount of an anti-PD-1antibody that does not competitively inhibit binding of PD-L1 or PD-L2to PD-1 expressed on the surface of the T cell.

In some embodiments, the present invention also provides a method forincreasing T cell effector function, comprising contacting a T cell withan anti-PD-1 antibody that does not competitively inhibit binding ofPD-L1 or PD-L2 to PD-1 expressed on the surface of the T cell.

Additionally, the present invention provides a method for increasinglymphocyte secretion of a cytokine selected from the group consisting ofIL-6, IL-12, IL-18, TNF-α, IL-1β and GM-CSF in a human patient in needof increased T cell effector function, comprising administering to thepatient a therapeutically effective amount of an anti-PD-1 antibody thatdoes not competitively inhibit binding of PD-L1 or PD-L2 to PD-1expressed on the surface of a T cell.

The present invention provides a method of treating cancer in a mammal,comprising contacting a T cell in a mammal in need thereof with acombination of: (a) an effective amount of an anti-PD-1 antibody thatcompetitively inhibits binding of PD-L1 or PD-L2 to PD-1 expressed onthe surface of the T cell; and (b) an effective amount of an anti-PD-1antibody that does not competitively inhibit binding of PD-L1 or PD-L2to PD-1 expressed on the surface of the T cell.

The present invention provides a method of producing anti-PD-1antibodies comprising a HCVR, a LCVR, or a combination thereof.Accordingly, also provided herein is an isolated nucleic acid comprisinga nucleotide sequence encoding the HCVR and/or LCVR of the presentdisclosure, as well as a host cell comprising an isolated nucleic acidof the invention.

The antibodies of the present invention can also be used in diagnostictesting. For example, the invention provides a method of diagnosing aPD-1-mediated disease or disorder, e.g., adaptive immune resistance, ina patient who has cancer.

These and other aspects and advantages of the invention will becomeapparent upon consideration of the following figures, detaileddescription and claims. As used herein, “including” means withoutlimitation, and the examples cited are non-limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1 is an alignment of the sequences of 26 heavy chain variableregions (HCVR) from antibodies that bind to human PD-1. The lowersegment of the sequences is a continuation of the upper segment.Framework regions FR1 through FR4 are indicated. Complementaritydetermining regions CDR1 through CDR3 are also shown.

FIG. 2 is an alignment of the sequences of 27 light chain variableregions (LCVR) from antibodies that bind to human PD-1. The lowersegment of the sequences is a continuation of the upper segment.Framework regions FR1 through FR4 are indicated. Complementaritydetermining regions CDR1 through CDR3 are also shown.

FIG. 3 is a bar graph summarizing data on binding of 32 mouseanti-human-PD-1 antibodies to PD-1-HIS. The specific HCVR and LCVRpairings for the 32 antibodies are indicated in the boxed text. Bindingwas evaluated by ELISA. Data represent average of two experiments.

FIG. 4 is a bar graph summarizing results from assays to measure theeffectiveness of anti-PD-1 antibodies in relieving PD-L1 dependentinhibition of activation of human peripheral blood mononuclear cells(PBMCs). Treatments of cells were carried out in 96-well plates for 3-5days. All treatments included plate-bound CD3 and soluble CD28 in PBS.Cells were treated additionally with nothing (positive control); withPD-L1 alone; or with PD-L1 plus an anti-PD-L1 antibody (EH12.2H7,pembrolizumab, 246A10 or 244C8). At the end of the treatment period,cells were transferred to a microwell array for IFNγ determination atthe level of individual cells, with sample cell populations in the rangeof approximately 50-100 cells.

FIG. 5 is a bar graph summarizing the results of differential T cellactivation in response to PD-1 blockade. These results indicate thatantibodies 388D4, 413E1, 246A10 and 244C8 elicited similar secretionlevels or enhanced secretion levels of IFNγ and TNFα as compared toantibody EH12.2H7 or pembrolizumab, under conditions of suboptimalactivation (achieved by the treatment with anti-CD3 and anti-CD28),which may mimic activation conditions that occur in vivo.

FIG. 6 is a bar graph summarizing the results of antigen recall assaysusing cytomegalovirus in human PBMC. These results indicate thatanti-PD-1 antibodies 246A10, 244C8, 413D2, 388D4, and 413E1 induceincreased levels of IFNγ compared to antibody isotype controls.

FIGS. 7A and 7B summarize the results of mixed lymphocyte reactionassays using anti-PD-1 antibodies and human PBMCs. Humanized versions ofclone 388D4 (“D4-HC3+LC1”; “D4-HC1+LC3”; and “D4-HC3+LC3”) appear toinduce cytokine release (FIG. 7A) and CD25 upregulation (FIG. 7B)similar to nivolumab. Humanized versions of clone 244C8 (“C8-HC1+LC1”;“C8-HC1+LC3”; and “C8-HC2+LC1”) appear to induce increased levels ofcytokine release (IFNγ) compared with 388D4 or nivolumab (FIG. 7A). Tcells incubated with 244C8 also appear to exhibit a higher degree ofactivation, as inferred from CD25 expression (FIG. 7B).

FIG. 8 indicates regions within the PD-1 amino acid sequence (SEQ IDNO:97) bound by certain antibodies of the present invention (246A10,244C8, 388D4, 413D2, and 413E1), as determined by peptide mapping. ThePD-1 amino acid sequences corresponding to the sequences of inhibitorypeptides are underlined.

FIGS. 9A-9D show selectivity of anti-PD-1 antibodies for the PD-1extracellular domain over other immunomodulatory cell surface proteinssuch as ICOS (inducible T-cell costimulator) (FIG. 9A), CD28 (FIG. 9C),or CTLA4 (FIG. 9D). Binding of anti-PD-1 antibodies to PD-1 is shown inFIG. 9B (EH12.2H7 is an anti-PD-1 antibody commercially available as alaboratory reagent). Anti-PD-1 antibodies 388D4(100388_D4VH3_100389_D4VK5 in Table 3), 413E1 (100413_E1VH9_100414_E1VK5in Table 3), 244C8, and 246A10 were tested.

FIGS. 10A and 10B are amino acid sequence alignments of humanized andmouse antibody sequences. FIG. 10A shows alignments for 100388_D4_VH3(mouse) with humanized heavy chain variable regions (100388_D4_HC1;100388_D4_HC2; and 100388_D4_HC3) and 100389_D4_VK5 (mouse) withhumanized light chain variable regions (100389_D4_LC1; 100389_D4_LC2;and 100389_D4_LC3). FIG. 10B shows alignments for 100244_C8_VH3 (mouse)with humanized heavy chain variable regions (100244_C8_HC1;100244_C8_HC2; and 100244_C8_HC3) and 100245_C8_VK5m1 (mouse) withhumanized light chain variable regions (100245_C8_LC1; 100245_C8_LC2;and 100245_C8_LC3).

FIGS. 11A-11D are flow cytometry histogram plots showing that antibody388D4 blocks binding of soluble PD-L1 to HEK293 cells expressing PD-1,while antibody 244C8 does not. HEK293 cells expressing PD-1 wereincubated with 10 μg/m1 of an isotype antibody (negative control),commercially available antibody EH12.2H7 (positive control) antibody388D4, or antibody 244C8. Cells were washed and stained with solublePD-L1-Ig protein fluorescently labeled with Alexa-488. Cells were washedagain, and PD-L1 binding (by displacing previously bound antibody) wasassessed by conventional fluorescence activated cell sorting (FACS)analysis. FIG. 11A shows data from FACS analysis of anti-PD-1 antibodyEH12.2H7 (positive control); FIG. 11B shows data from FACS analysis ofanti-PD-1 antibody mIgG1K (negative control); FIG. 11C shows data fromFACS analysis of anti-PD-1 antibody 388D4; FIG. 11D shows data from FACSanalysis of anti-PD-1 antibody 244C8.

FIG. 12 is a histogram showing restoration of T cell function by PD-1blockade with nivolumab or different humanized forms of antibodies 388D4and 244C8, i.e., 388D4-2, 388D4-3, 244C8-1, 244C8-2, and 244C8-3. Apopulation of 3×10⁵ dissociated and suspended human cells from anon-small cell lung cancer (NSCLC) biopsy, which included 17%lymphocytes (activated as described above) was incubated for 24 hourswith anti-PD-1 antibodies at a concentration of 20 μg/mL. IFNγ wasmeasured by ELISA, and the data are expressed in terms offold-activation relative to treatment with the isotype control antibody.Each of the anti-PD-1 antibodies restored T cell function, increasingIFNγ secretion approximately 7-fold to 7.5 fold, relative to the isotypecontrol.

FIG. 13 is a histogram summarizing results from an experiment to measurethe increase in T cell effector function, as indicated by IFNγsecretion, in response to treatment with antibody 244C8-2 alone versustreatment with 244C8-2 plus 388D4-2, with results normalized relative tothe response to treatment with nivolumab. A population of 3×10⁵ cells,which included 7.5% lymphocytes sub-optimally activated as describedabove, was incubated for 24 hours with anti-PD-1 antibodies at a totalantibody concentration of 20 μg/mL. As shown in FIG. 13, treatment with244C8-2 alone increased IFNγ 1.77-fold (±0.19 sd), while treatment with244C8-2 in combination with 388D4-2 increased IFNγ secretion 2.11-fold(±0.21 sd).

FIG. 14 is a histogram summarizing results from an experiment showingthat treatment with the combination of nivolumab and antibody 244C8-2resulted in greater restoration of T cell effector function thantreatment with nivolumab alone, antibody 244C8-2 alone, or antibody388D4-2 alone. In each treatment, a population of 3×10⁵ cells, whichincluded 9% lymphocytes (sub-optimally activated as described above) wasincubated for 24 hours with anti-PD-1 antibodies at a totalconcentration of 20 μg/mL. Following PD-1 blockade, cells andsupernatants were collected for ELISA measurement of IFNγ. Data areexpressed in terms of fold-induction of IFNγ secretion, relative totreatment with the isotype control antibody.

FIGS. 15A-15F are histograms summarizing the results of a mixedlymphocyte reaction (MLR) assay performed on human PBMCs treated withanti-PD-1 antibodies. The MLR assay was performed using commerciallyavailable monocyte-derived dendritic cells as stimulator cells andpurified CD4+ T lymphocytes as responder cells from a different healthyblood donor. Supernatants were collected 2.5 days after beginning theassay. Treatment with antibody 244C8-1 (100244_C8_HC1+100245_C8_LC1)resulted in increased secretion of cytokines IL-6, IL-12, IL-18, TNF-α,GM-CSF, and IL-1β, in comparison with antibody 388D4-2(100388_D4_HC3+100389_D4_LC3) or an IgG4 isotype control. (FIG. 15A,IL-6; FIG. 15B, IL-12; FIG. 15C, IL-18; FIG. 15D TNF-α; FIG. 15E, IL-1β;FIG. 15F, GM-CSF)

FIGS. 16A-16F are histograms summarizing the results of an experimentshowing alteration of tumor infiltrating lymphocyte (TIL) function byPD-1 blockade with anti-PD-1 antibodies 388D4-2 and 244C8-2. Apopulation of 3×10⁵ dissociated and suspended human cells from anon-small cell lung cancer (NSCLC) biopsy, which included 7% stimulated,tumor-infiltrating lymphocytes was incubated for 24 hours with anti-CD3and anti-CD28 antibodies along with an anti-PD-1 antibody or IgG4isotype control at a concentration of 10 μg/mL. Treatment with antibody244C8-2 (100244_C8_HC1+100245_C8_LC3) resulted in increased secretion ofcytokines IL-6, IL-12, IL-18, TNF-α, GM-CSF, and IL-1β, in comparisonwith antibody 388D4-2 (100388_D4_HC3+100389_D4_LC3) or the IgG4 isotypecontrol. (FIG. 16A, IL-6; FIG. 16B, IL-12; FIG. 16C, IL-18; FIG. 16DTNF-α; FIG. 16E, IL-1β; FIG. 16F, GM-CSF).

FIGS. 17A-17C show the results from an in vivo efficacy experimentinvolving patient-derived xenograft (PDX) lung tumor growth in humanizedmice treated with vehicle control, antibody 388D4-3, antibody 244C8-2,pembrolizumab, or a combination of antibody 244C8-2 and pembrolizumab.Animals received a total of six intra-peritoneal doses of antibody atfive-day intervals (Q5D×6) 5 mg/kg. In the treatment groups thatreceived 388D4-3, 244C8-2 or pembrolizumab, the first dose of theantibody was given as a 10 mg/kg dose, followed by the additional dosesat the 5 mg/kg dose. The combination treatment group received a dose ofeach 5 mg/kg of pembrolizumab and 5 mg/kg of 244C8-2 at each dosing timepoint. Tumor volumes were measured twice weekly (Day 3, 6, 10, 13, 17,20, 24 and 28) using a digital caliper to determine length and width ofthe tumors. All animals were sacrificed at day 28 after dosinginitiation. Error bars represent the 95% confidence interval (n=10). Alltreatment groups showed significant tumor growth inhibition compared tothe vehicle control group. As shown in FIG. 17A, no significantdifference in tumor growth inhibition was observed among treatment withantibody 388D4-3, antibody 244C8-2, pembrolizumab, or the combination ofantibody 244C8-2 with pembrolizumab. FIG. 17B is a boxplot of tumorvolumes for each treatment arm at day 28 (end of study) of theexperiment described in FIG. 17A. The tumor volume for each treatmentgroup was significantly smaller than that of the vehicle group. StudentT-test p values between each treatment group and vehicle group were:0.00167 (pembrolizumab), 0.00105 (388D4-3), 0.00277 (244C8-2), and0.00275 (pembrolizumab+244C8), respectively. FIG. 17C is a histogramshowing percentage tumor volume of each treatment group relative tovehicle on day 28 of the experiment described in FIGS. 17A and 17B. Thecalculated percent tumor growth inhibition (% TGI) for each treatment isshown above each bar.

DETAILED DESCRIPTION OF THE INVENTION

The anti-PD-1 antibodies disclosed herein are based on the antigenbinding sites of certain monoclonal antibodies selected on the basis ofbinding to human Programmed Death-1 (PD-1) protein (UniProt #Q15116).The antibodies contain immunoglobulin variable region CDR sequences thatdefine binding sites for human PD-1.

By virtue of the PD-1 signal blocking or PD-1 neutralizing activity ofcertain of these antibodies, they are useful for treating various typesof cancer, including inhibiting tumor growth. In some embodiments (e.g.,when used as therapeutic agents), the antibodies can be engineered tominimize or eliminate an immune response when administered to a humanpatient. Various features and aspects of the invention are discussed inmore detail below.

As used herein, “isolated antibody” means an antibody that issubstantially free of its natural environment. For instance, an isolatedantibody or nucleic acid is substantially free of cellular material andother proteins from the cell or tissue source from which it is derived.

As used herein, unless otherwise indicated, “antibody” means an intactantibody or antigen-binding fragment of an antibody, including an intactantibody or antigen-binding fragment that has been modified orengineered, or that is a human antibody. Examples of antibodies thathave been modified or engineered are chimeric antibodies, humanizedantibodies, multiparatopic antibodies (e.g., biparatopic antibodies),and multispecific antibodies (e.g., bispecific antibodies). Examples ofantigen-binding fragments include Fab, Fab′, F(ab′)₂, Fv, single chainantibodies (e.g., scFv), minibodies and diabodies.

The antibodies disclosed herein comprise: (a) an immunoglobulin heavychain variable region comprising the structureCDR_(H1)-CDR_(H2)-CDR_(H3), and (b) an immunoglobulin light chainvariable region comprising the structure CDR_(L1)-CDR_(L2)-CDR_(L3),wherein the heavy chain variable region and the light chain variableregion together define a single binding site for binding human PD-1protein.

In some embodiments, the isolated antibody that binds to PD-1 comprisesa heavy chain variable region (HCVR) having complementarity determiningregions (CDRs) selected from the group consisting of: CDRs 1-3 of SEQ IDNO: 1; CDRs 1-3 of SEQ ID NO: 2; CDRs 1-3 of SEQ ID NO: 3; CDRs 1-3 ofSEQ ID NO: 4; CDRs 1-3 of SEQ ID NO: 5; CDRs 1-3 of SEQ ID NO: 6; CDRs1-3 of SEQ ID NO: 7; CDRs 1-3 of SEQ ID NO: 8; CDRs 1-3 of SEQ ID NO: 9;CDRs 1-3 of SEQ ID NO: 10; CDRs 1-3 of SEQ ID NO: 11; CDRs 1-3 of SEQ IDNO: 12; CDRs 1-3 of SEQ ID NO: 13; CDRs 1-3 of SEQ ID NO: 14; CDRs 1-3of SEQ ID NO: 15; CDRs 1-3 of SEQ ID NO: 16; CDRs 1-3 of SEQ ID NO: 17;CDRs 1-3 of SEQ ID NO: 18; CDRs 1-3 of SEQ ID NO: 19; CDRs 1-3 of SEQ IDNO: 20; CDRs 1-3 of SEQ ID NO: 21; CDRs 1-3 of SEQ ID NO: 22; CDRs 1-3of SEQ ID NO: 23; CDRs 1-3 of SEQ ID NO: 24; CDRs 1-3 of SEQ ID NO: 25;and CDRs 1-3 of SEQ ID NO: 26.

In some embodiments, the isolated antibody that binds to PD-1 comprisesa HCVR selected from the group consisting of SEQ ID NOs: 1-26.

In some embodiments, the isolated antibody that binds to PD-1 comprisesa light chain variable region (LCVR) having CDRs selected from the groupconsisting of: CDRs 1-3 of SEQ ID NO: 27; CDRs 1-3 of SEQ ID NO: 28;CDRs 1-3 of SEQ ID NO: 29; CDRs 1-3 of SEQ ID NO: 30; CDRs 1-3 of SEQ IDNO: 31; CDRs 1-3 of SEQ ID NO: 32; CDRs 1-3 of SEQ ID NO: 33; CDRs 1-3of SEQ ID NO: 34; CDRs 1-3 of SEQ ID NO: 35; CDRs 1-3 of SEQ ID NO: 36;CDRs 1-3 of SEQ ID NO: 37; CDRs 1-3 of SEQ ID NO: 38; CDRs 1-3 of SEQ IDNO: 39; CDRs 1-3 of SEQ ID NO: 40; CDRs 1-3 of SEQ ID NO: 41; CDRs 1-3of SEQ ID NO: 42; CDRs 1-3 of SEQ ID NO: 43; CDRs 1-3 of SEQ ID NO: 44;CDRs 1-3 of SEQ ID NO: 45; CDRs 1-3 of SEQ ID NO: 46; CDRs 1-3 of SEQ IDNO: 47; CDRs 1-3 of SEQ ID NO: 48; CDRs 1-3 of SEQ ID NO: 49; CDRs 1-3of SEQ ID NO: 50; CDRs 1-3 of SEQ ID NO: 51; CDRs 1-3 of SEQ ID NO: 52;and CDRs 1-3 of SEQ ID NO: 53.

In some embodiments, the isolated antibody that binds to PD-1 comprisesa LCVR selected from the group consisting of SEQ ID NOs: 27-53.

In some embodiments, the isolated antibody that binds to PD-1 comprisesa HCVR selected from the group consisting of SEQ ID NOs: 1-26 and a LCVRselected from the group consisting of SEQ ID NOs: 27-53. Examples ofpairings of HCVR and LCVR are provided throughout the presentdisclosure, but additional functional pairings are within the scope ofthe invention.

In some embodiments, the antibody comprises a HCVR having the sequenceset forth in SEQ ID NO: 4 and a LCVR having the sequence set forth inSEQ ID NO: 28 (designated as 244C7 in Table 3); a HCVR having thesequence set forth in SEQ ID NO: 4 and a LCVR having the sequence setforth in SEQ ID NO: 27 (244C7m1); a HCVR having the sequence set forthin SEQ ID NO: 1 and a LCVR having the sequence set forth in SEQ ID NO:28 (244C8); a HCVR having the sequence set forth in SEQ ID NO: 1 and aLCVR having the sequence set forth in SEQ ID NO: 27 (244C8m1); a HCVRhaving the sequence set forth in SEQ ID NO: 3 and a LCVR having thesequence set forth in SEQ ID NO: 31 (246F7); a HCVR having the sequenceset forth in SEQ ID NO: 5 and a LCVR having the sequence set forth inSEQ ID NO: 44 (258C1); a HCVR having the sequence set forth in SEQ IDNO: 2 and a LCVR having the sequence set forth in SEQ ID NO: 30 (258F6);a HCVR having the sequence set forth in SEQ ID NO: 2 and a LCVR havingthe sequence set forth in SEQ ID NO: 29 (258F6m); a HCVR having thesequence set forth in SEQ ID NO: 6 and a LCVR having the sequence setforth in SEQ ID NO: 34 (392C4); a HCVR having the sequence set forth inSEQ ID NO: 7 and a LCVR having the sequence set forth in SEQ ID NO: 41(394D5); a HCVR having the sequence set forth in SEQ ID NO: 8 and a LCVRhaving the sequence set forth in SEQ ID NO: 35 (394G1); a HCVR havingthe sequence set forth in SEQ ID NO: 12 and a LCVR having the sequenceset forth in SEQ ID NO: 39 (388C12A); a HCVR having the sequence setforth in SEQ ID NO: 12 and a LCVR having the sequence set forth in SEQID NO: 32 (388C12B); a HCVR having the sequence set forth in SEQ ID NO:13 and a LCVR having the sequence set forth in SEQ ID NO: 39 (388C16A);a HCVR having the sequence set forth in SEQ ID NO: 13 and a LCVR havingthe sequence set forth in SEQ ID NO: 32 (388C16B); a HCVR having thesequence set forth in SEQ ID NO: 9 and a LCVR having the sequence setforth in SEQ ID NO: 38 (392C5A); a HCVR having the sequence set forth inSEQ ID NO: 9 and a LCVR having the sequence set forth in SEQ ID NO: 37(392C5B); a HCVR having the sequence set forth in SEQ ID NO: 17 and aLCVR having the sequence set forth in SEQ ID NO: 40 (392D2); a HCVRhaving the sequence set forth in SEQ ID NO: 16 and a LCVR having thesequence set forth in SEQ ID NO: 43 (392H4); a HCVR having the sequenceset forth in SEQ ID NO: 20 and a LCVR having the sequence set forth inSEQ ID NO: 53 (246A10); a HCVR having the sequence set forth in SEQ IDNO: 18 and a LCVR having the sequence set forth in SEQ ID NO: 47(388D4); a HCVR having the sequence set forth in SEQ ID NO: 19 and aLCVR having the sequence set forth in SEQ ID NO: 48 (392A6); a HCVRhaving the sequence set forth in SEQ ID NO: 21 and a LCVR having thesequence set forth in SEQ ID NO: 52 (411C2); a HCVR having the sequenceset forth in SEQ ID NO: 22 and a LCVR having the sequence set forth inSEQ ID NO: 51 (413D2); or a HCVR having the sequence set forth in SEQ IDNO: 25 and a LCVR having the sequence set forth in SEQ ID NO: 45(413E1).

In some embodiments, the antibody comprises a HCVR having the sequenceset forth in SEQ ID NO: 20 and a LCVR having the sequence set forth inSEQ ID NO: 53 (246A10); a HCVR having the sequence set forth in SEQ IDNO: 25 and a LCVR having the sequence set forth in SEQ ID NO: 45(413E1); a HCVR having the sequence set forth in SEQ ID NO: 22 and aLCVR having the sequence set forth in SEQ ID NO: 51 (413D2); a HCVRhaving the sequence set forth in SEQ ID NO: 18 and a LCVR having thesequence set forth in SEQ ID NO: 47 (388D4); a HCVR having the sequenceset forth in SEQ ID NO: 1 and a LCVR having the sequence set forth inSEQ ID NO: 28 (244C8); or a HCVR having the sequence set forth in SEQ IDNO: 9 and a LCVR having the sequence set forth in SEQ ID NO: 38(392C5A).

In some embodiments, the isolated antibody that binds to PD-1 binds to asequence in PD-1 selected from the group consisting of SEQ ID NO: 54,SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ IDNO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73,SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ IDNO: 83, and SEQ ID NO: 84.

As used herein, an “antibody that binds to PD-1, comprising” a HCVR orLCVR, means an antibody comprising the HCVR or LCVR, as opposed to aPD-1 protein comprising the HCVR or LCVR.

In some embodiments, the antibody binds specifically to PD-1. This meansthat the antibody binds to PD-1 protein in a sample, with negligiblebinding to other proteins present in the sample, under a given set ofbinding reaction conditions.

Examples of antibody fragments include, a Fab, Fab′, F(ab′)₂, Fv, scFv,dAb, and a diabody.

A “Fab fragment” comprises one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the CH2 andCH3 domains of an antibody. The two heavy chain fragments are heldtogether by two or more disulfide bonds and by hydrophobic interactionsof the CH3 domains.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the VH domain and the CH1 domain and also the regionbetween the CH1 and CH2 domains, such that an interchain disulfide bondcan be formed between the two heavy chains of two Fab′ fragments to forma F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 and C_(H)² domains, such that an interchain disulfide bond is formed between thetwo heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

A “single-chain Fv antibody” (or “scFv antibody”) refers to antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Pluckthun (1994) The Pharmacology OfMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315. See also, PCT Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

A “diabody” is a small antibody fragment with two antigen-binding sites.The fragments comprise a heavy chain variable region (VH) connected to alight chain variable region (VL) in the same polypeptide chain (VH-VL orVL-VH). By using a linker that is too short to allow pairing between thetwo domains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. Diabodies are described in, e.g., patent documents EP 404,097; WO93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

A “domain antibody fragment” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore VH regions are covalently joined with a peptide linker to create abivalent domain antibody fragment. The two VH regions of a bivalentdomain antibody fragment may target the same or different antigens.

In some embodiments, the antibody is modified or engineered. Examples ofmodified or engineered antibodies include chimeric antibodies,multiparatopic antibodies (e.g., biparatopic antibodies), andmultispecific antibodies (e.g., bispecific antibodies).

As used herein, “multiparatopic antibody” means an antibody thatcomprises at least two single domain antibodies, in which at least onesingle domain antibody is directed against a first antigenic determinanton an antigen and at least one other single domain antibody is directedagainst a second antigenic determinant on the same antigen. Thus, forexample, a “biparatopic” antibody comprises at least one single domainantibody directed against a first antigenic determinant on an antigenand at least one further single domain antibody directed against asecond antigenic determinant on the same antigen.

As used herein, “multispecific antibody” means an antibody thatcomprises at least two single domain antibodies, in which at least onesingle domain antibody is directed against a first antigen and at leastone other single domain antibody is directed against a second antigen(different from the first antigen). Thus, for example, a “bispecific”antibody is one that comprises at least one single domain antibodydirected against a first antigen and at least one further single domainantibody directed against a second antigen, e.g., different from thefirst antigen.

In some embodiments, the antibodies disclosed herein are monoclonalantibodies, e.g., murine monoclonal antibodies. Methods of producingmonoclonal antibodies are known in the art. See, for example, Pluckthun(1994) The Pharmacology Of Monoclonal Antibodies, Vol. 113, Rosenburgand Moore eds. Springer-Verlag, New York, pp. 269-315.

In some embodiments, antibodies are modified to reduce immunogenicity.When the antibodies are to be administered to a human, the antibodiescan be “humanized” to reduce or eliminate antigenicity in humans.Accordingly, in some embodiments, the antibody comprises a humanized orhuman framework region (FR).

In some embodiments, the isolated antibody that binds to PD-1 comprisesa HCVR selected from the group consisting of SEQ ID NOs: 85-90.

In some embodiments, the isolated antibody that binds to PD-1 comprisesa LCVR selected from the group consisting of SEQ ID NOs: 91-96.

In certain embodiments, the isolated antibody that binds to PD-1comprises a HCVR selected from the group consisting of SEQ ID NOs: 85-90and a LCVR selected from the group consisting of SEQ ID NOs: 91-96.Examples of pairings of HCVRs and LCVRs are provided throughout thepresent disclosure, but additional functional pairings are within thescope of the invention.

In some embodiments, the isolated antibody comprises a HCVR having thesequence set forth in SEQ ID NO: 90 and a LCVR having the sequence setforth in SEQ ID NO: 94; a HCVR having the sequence set forth in SEQ IDNO: 88 and a LCVR having the sequence set forth in SEQ ID NO: 96; a HCVRhaving the sequence set forth in SEQ ID NO: 90 and a LCVR having thesequence set forth in SEQ ID NO: 96; a HCVR having the sequence setforth in SEQ ID NO: 85 and a LCVR having the sequence set forth in SEQID NO: 91; a HCVR having the sequence set forth in SEQ ID NO: 85 and aLCVR having the sequence set forth in SEQ ID NO: 93; or a HCVR havingthe sequence set forth in SEQ ID NO: 86 and a LCVR having the sequenceset forth in SEQ ID NO: 91.

Methods for reducing or eliminating the antigenicity of antibodies andantibody fragments are known in the art. In one approach, a nucleic acidencoding a PD-1 antibody disclosed herein is modified, for example, byreplacing the mouse constant region with human heavy- and light-chainconstant regions (e.g., U.S. Pat. No. 4,816,567; Morrison, et al., 1984,Proc. Natl. Acad. Sci. USA, 81:6851) to produce what is commonlyreferred to as a chimeric antibody.

A humanized antibody generally has one or more amino acid residues froma source that is non-human. The non-human amino acid residues are oftenreferred to as “import” residues, and are typically taken from an“import” variable domain. Humanization can be performed generallyfollowing the method of Winter and co-workers (Jones et al., 1986,Nature 321:522-525; Riechmann et al., 1988, Nature, 332:323-327;Verhoeyen et al., 1988, Science 239:1534-1536), by substitutingnon-human CDRs or CDR sequences for the corresponding sequences of ahuman antibody. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in non-human, for example,murine antibodies. Preferably, a humanized antibody has the same orsubstantially the same affinity for the antigen as the non-human, e.g.,mouse antibody from which it was derived.

In an approach known as CDR grafting, the CDRs of the light and heavychain variable regions are grafted into frameworks from another species.For example, murine CDRs can be grafted into human FRs. In someembodiments, the CDRs of the light and heavy chain variable regions of aPD-1 antibody are grafted into human FRs or consensus human FRs. Tocreate consensus human FRs, FRs from several human heavy chain or lightchain amino acid sequences are aligned to identify a consensus aminoacid sequence. CDR grafting is described in, e.g., U.S. Pat. No.7,022,500 (Queen); U.S. Pat. No. 6,982,321 (Winter); U.S. Pat. No.6,180,370 (Queen); U.S. Pat. No. 6,054,297 (Carter); U.S. Pat. No.5,693,762 (Queen); U.S. Pat. No. 5,859,205 (Adair); U.S. Pat. No.5,693,761 (Queen); U.S. Pat. No. 5,565,332 (Hoogenboom); U.S. Pat. No.5,585,089 (Queen); U.S. Pat. No. 5,530,101 (Queen); Jones et al. (1986)Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327;Verhoeyen et al. (1988) Science 239: 1534-1536; and Winter (1998) FEBSLett 430: 92-94.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicity.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequencethat is closest to that of the murine is then accepted as the FR for thehumanized antibody (Sims et al., 1987, J. Immunol. 151:2296; Chothia etal., 1987, J Mol. Biol. 196:901). Another method uses a particularframework derived from the consensus sequence of all human antibodies ofa particular subgroup of light or heavy chains. The same framework maybe used for several different humanized antibodies (Carter et al., 1992,Proc. Natl. Acad. Sci. USA 89:4285; Presta et al., 1993, J. Immunol.151:2623).

It is important for humanized antibodies to retain affinity for theantigen and other desirable biological properties. To achieve thisresult, humanized antibodies can be designed analyzing parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs that illustrateand display probable three-dimensional conformational structures ofselected candidate immunoglobulin sequences are available. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved.

Other methods to reduce immunogenicity include “reshaping,”“hyperchimerization,” and “veneering/resurfacing.” See, e.g., Vaswami etal., 1998, Ann. Allergy & Immunol. 81:105; Roguska et al., 1996, Prot.Engineer. 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman). In theveneering/resurfacing approach, the surface accessible amino acidresidues in the murine antibody are replaced by amino acid residues morefrequently found at the same positions in a human antibody. This type ofantibody resurfacing is described, e.g., in U.S. Pat. No. 5,639,641(Pedersen).

Another approach for converting a mouse antibody into a form suitablefor medical use in humans is known as ACTIVMAB™ technology (Vaccinex,Inc., Rochester, N.Y.), which involves use of a vaccinia virus-basedvector to express antibodies in mammalian cells. High levels ofcombinatorial diversity of IgG heavy and light chains are said to beproduced. See, e.g., U.S. Pat. No. 6,706,477 (Zauderer); U.S. Pat. No.6,800,442 (Zauderer); and U.S. Pat. No. 6,872,518 (Zauderer).

Another approach for converting a mouse antibody into a form suitablefor use in humans is technology practiced commercially by KaloBiosPharmaceuticals, Inc. (Palo Alto, Calif.). This technology involves theuse of a proprietary human “acceptor” library to produce an “epitopefocused” library for antibody selection.

Another approach for modifying a mouse antibody into a form suitable formedical use in humans is HUMAN ENGINEERING™ technology, which ispracticed commercially by XOMA (US) LLC. See, e.g., PCT Publication No.WO 93/11794 and U.S. Pat. No. 5,766,886 (Studnicka); U.S. Pat. No.5,770,196 (Studnicka); U.S. Pat. No. 5,821,123 (Studnicka); and U.S.Pat. No. 5,869,619 (Studnicka).

Humanization of antibodies is routine protein engineering. Nearly allmurine antibodies can be humanized by CDR grafting, resulting in theretention of antigen binding. See, e.g., Lo, Benny, K. C., editor, inAntibody Engineering: Methods and Protocols, Vol. 248, Humana Press, NewJersey, 2004.

In some embodiments, the antibodies are antagonists. As used herein,“antagonist” in reference to an anti-PD-1 antibody means an antibodythat inhibits the PD-1 signaling pathway in a cell (e.g., an immunecell). An antagonist anti-PD-1 antibody might inhibit the PD-1 signalingpathway by blocking the PD-1/PD-L1 or PD-1/PD-L2 interaction, but doesnot necessarily do so.

In some embodiments, the antibodies are agonists. As used herein,“agonist” in reference to an anti-PD-1 antibody means an antibody thatactivates the PD-1 signaling pathway in a cell (e.g., an immune cell).An agonist antibody might influence the PD-1/PD-L1 and/or PD-1/PD-L2interaction, but does not necessarily do so.

An antibody that binds to PD-1 and competitively inhibits the binding ofan antibody that contains one or more sequences disclosed herein iswithin the scope of the invention. In certain embodiments, the antibodycompetitively inhibits the binding of the antibody that comprises a HCVRhaving the sequence set forth in SEQ ID NO: 20 and a LCVR having thesequence set forth in SEQ ID NO: 53 (246A10); a HCVR having the sequenceset forth in SEQ ID NO: 25 and a LCVR having the sequence set forth inSEQ ID NO: 45 (413E1); a HCVR having the sequence set forth in SEQ IDNO: 22 and a LCVR having the sequence set forth in SEQ ID NO: 51(413D2); a HCVR having the sequence set forth in SEQ ID NO: 18 and aLCVR having the sequence set forth in SEQ ID NO: 47 (388D4); a HCVRhaving the sequence set forth in SEQ ID NO: 1 and a LCVR having thesequence set forth in SEQ ID NO: 28 (244C8). In some embodiments, theantibody also binds to a sequence in PD-1 selected from the groupconsisting of SEQ ID NOs: 54-84.

Methods for determining whether two or more antibodies compete forbinding to the same target are known in the art. For example, acompetitive binding, or competition, assay can be used to determinewhether one antibody blocks the binding of another antibody to thetarget. Typically, a competition assay involves the use of purifiedtarget antigen (e.g., PD-1) bound to a solid substrate or expressed oncells, an unlabeled test binding molecule (e.g., a test anti-PD-1antibody), and a labeled reference binding molecule (e.g., an antibodydisclosed herein). Competitive inhibition is measured by determining theamount of label bound to the solid substrate or cells in the presence ofthe test molecule. Usually (but not necessarily) the molecule is presentin excess of at least two-fold. A test antibody competes with thereference antibody or ligand (e.g., PD-L1 or PD-L2) for specific bindingto the antigen if an excess of one antibody inhibits binding of theother antibody or ligand by at least 50%, as measured in a competitionassay.

In an exemplary competition assay, a reference anti-PD-1 antibody (e.g.,an antibody disclosed herein) is biotinylated using commerciallyavailable reagents. The biotinylated reference antibody is mixed withserial dilutions of the test antibody or unlabeled reference antibody(self-competition control) resulting in a mixture of various molarratios of test antibody (or unlabeled reference antibody) to labeledreference antibody. The antibody mixture is added to a PD-1 coated-ELISAplate. The plate is then washed, and horseradish peroxidase(HRP)-strepavidin is added to the plate as the detection reagent. Theamount of labeled reference antibody bound to the target antigen isdetected following addition of a chromogenic substrate (e.g., TMB(3,3′,5,5′-tetramethylbenzidine) or ABTS(2,2″-azino-di-(3-ethylbenzthiazoline-6-sulfonate)), which are known inthe art. Optical density readings (OD units) are measured using aspectrophotometer. OD units corresponding to zero percent inhibition aredetermined from wells without any competing antibody. OD unitscorresponding to 100% inhibition, i.e., the assay background, aredetermined from wells without any labeled reference antibody or testantibody. Percent inhibition of labeled reference antibody to PD-1 bythe test antibody (or the unlabeled reference antibody) at eachconcentration is calculated as follows: % inhibition=(1−(OD units−100%inhibition)/(0% inhibition−100% inhibition))*100. Persons skilled in theart will appreciate that the competition assay can be performed usingvarious detection systems known in the art.

Antibodies identified by competition assay (e.g., competing antibodies)include antibodies binding to the same epitope, or similar (e.g.,overlapping) epitopes, as the reference antibody. In addition, thecompetition assay can identify antibodies binding to an adjacent epitopesufficiently proximal to the epitope bound by the reference antibody forsteric hindrance to occur.

Two antibodies bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody to the antigen reduce or eliminate binding of the other. Twoantibodies bind to overlapping epitopes if only a subset of the aminoacid mutations that reduce or eliminate binding of one antibody to theantigen reduce or eliminate binding of the other.

A competition assay may be conducted in both directions to ensure thatthe presence of the label does not interfere with or otherwise inhibitbinding. For example, in the first direction, the reference antibody islabeled and the test antibody is unlabeled, and in the second direction,the test antibody is labeled and the reference antibody is unlabeled.

In certain embodiments, the present invention provides a method forincreasing T cell effector function, comprising contacting a T cell witha combination of: (a) an effective amount of an anti-PD-1 antibody thatcompetitively inhibits binding of PD-L1 or PD-L2 to PD-1 expressed onthe surface of the T cell; and (b) an effective amount of an anti-PD-1antibody that does not competitively inhibit binding of PD-L1 or PD-L2to PD-1 expressed on the surface of the T cell. In some embodiments,increasing T cell effector function includes, e.g., increased secretionof effector cytokines, as demonstrated herein.

In some embodiments, the T cell is contacted with the combination ofantibodies in vivo. For example, in certain embodiments, the T cell iscontacted with the combination in a human patient in need of increased Tcell effector function.

In some embodiments, the present invention also provides a method forincreasing T cell effector function, comprising contacting a T cell withan anti-PD-1 antibody that does not competitively inhibit binding ofPD-L1 or PD-L2 to PD-1 expressed on the surface of the T cell. Incertain embodiments, the T cell is contacted with an antibody thatcomprises a heavy chain variable region having complementaritydetermining regions (CDRs) selected from the group consisting of CDRs1-3 of SEQ ID NO:85, CDRs 1-3 of SEQ ID NO:86, and CDRs 1-3 of SEQ IDNO:87; and a light chain variable region having CDRs selected from thegroup consisting of CDRs 1-3 of SEQ ID NO:91, CDRs 1-3 of SEQ ID NO:92,and CDRs 1-3 of SEQ ID NO:93. In some embodiments, the T cell iscontacted with an antibody that comprises a heavy chain variable regionselected from the group consisting of SEQ ID NOS: 85, 86 and 87 and alight chain variable region selected from the group consisting of SEQ IDNOS: 91, 92 and 93. In certain embodiments, the T cell is contacted withan antibody that is selected from the group consisting of antibody244C8-1, antibody 244C8-2 and antibody 244C8-3.

In some embodiments, the present invention provides a method forincreasing lymphocyte secretion of a cytokine selected from the groupconsisting of IL-6, IL-12, IL-18, TNF-α, IL-1β and GM-CSF in a humanpatient in need of increased T cell effector function, comprisingadministering to the patient a therapeutically effective amount of ananti-PD-1 antibody that does not competitively inhibit binding of PD-L1or PD-L2 to PD-1 expressed on the surface of a T cell. In certainembodiments, the patient is administered an antibody that comprises aheavy chain variable region having complementarity determining regions(CDRs) selected from the group consisting of CDRs 1-3 of SEQ ID NO:85,CDRs 1-3 of SEQ ID NO:86, and CDRs 1-3 of SEQ ID NO:87; and a lightchain variable region having CDRs selected from the group consisting ofCDRs 1-3 of SEQ ID NO:91, CDRs 1-3 of SEQ ID NO:92, and CDRs 1-3 of SEQID NO:93. In some embodiments, the patient is administered an antibodythat comprises a heavy chain variable region selected from the groupconsisting of SEQ ID NOS: 85, 86 and 87 and a light chain variableregion selected from the group consisting of SEQ ID NOS: 91, 92 and 93.In certain embodiments, the patient is administered an antibody that isselected from the group consisting of antibody 244C8-1, antibody 244C8-2and antibody 244C8-3.

In some embodiments, the present invention also provides a method oftreating cancer in a mammal, comprising contacting a T cell in a mammalin need thereof with a combination of: (a) an effective amount of ananti-PD-1 antibody that competitively inhibits binding of PD-L1 or PD-L2to PD-1 expressed on the surface of the T cell; and (b) an effectiveamount of an anti-PD-1 antibody that does not competitively inhibitbinding of PD-L1 or PD-L2 to PD-1 expressed on the surface of the Tcell.

The presently disclosed method of treating cancer with a combination ofanti-PD-1 antibodies can be used to treat various cancers. In someembodiments, the cancer is selected from the group consisting of:melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma,breast cancer, colon cancer, lung cancer, esophageal cancer, squamouscell carcinoma of the head and neck, liver cancer, ovarian cancer,cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, andlymphoma.

In some embodiments, the anti-PD-1 antibody that competitively inhibitsbinding of PD-L1 or PD-L2 to PD-1 expressed on the surface of a T cellis selected from the group consisting of: 388D4, nivolumab,pembrolizumab, EH12.2H7 and J105. In some embodiments, the anti-PD-1antibody that competitively inhibits binding of PD-L1 or PD-L2 to PD-1expressed on the surface of a T cell is 388D4.

In some embodiments, the anti-PD-1 antibody that does not competitivelyinhibit binding of PD-L1 or PD-L2 to PD-1 expressed on the surface of aT cell is 244C8. In some embodiments, the anti-PD-1 antibody that doesnot competitively inhibit binding of PD-L1 or PD-L2 to PD-1 expressed onthe surface of the T cell binds to one or more of the following aminoacid sequences: SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO:83 and SEQ ID NO: 84. In some embodiments, the anti-PD-1 antibody bindsto all of the following amino acid sequences: SEQ ID NO: 74, SEQ ID NO:77, SEQ ID NO: 80, SEQ ID NO: 83 and SEQ ID NO: 84. In some embodiments,the anti-PD-1 antibody that does not competitively inhibit binding ofPD-L1 or PD-L2 to PD-1 expressed on the surface of the T cell binds to aPD-1 epitope bound by 244C8. In some embodiments, the anti-PD-1 antibodythat does not competitively inhibit binding of PD-L1 or PD-L2 to PD-1expressed on the surface of the T cell competes with 244C8 for bindingto PD-1.

The presently disclosed method of treating cancer with an anti-PD-1antibody that competitively inhibits binding of PD-L1 or PD-L2 to PD-1expressed on the surface of T cells, and an effective amount of ananti-PD-1 antibody that does not competitively inhibit binding of PD-L1or PD-L2 to PD-1 expressed on the surface of the T cells, increases Tcell effector function to a greater extent than an equivalent amount ofeither anti-PD-1 antibody alone. In some embodiments, the combinationyields an additive effect on T cell effector function. In someembodiments, the combination yields a synergistic effect on T celleffector function.

The present invention provides isolated nucleic acids comprising anucleotide sequence encoding a HCVR and/or a LCVR disclosed herein, or afragment thereof. A nucleic acid according to the present invention maycomprise DNA or RNA, and may be wholly or partially synthetic. Forexample, DNA molecules encoding an HCVR and/or LCVR disclosed herein canbe chemically synthesized. Synthetic DNA molecules can be ligated toother appropriate nucleotide sequences, including, e.g., constant regioncoding sequences, and expression control sequences, to produceconventional gene expression constructs encoding the desired antibodies.Production of defined gene constructs is within routine skill in theart. Alternatively, nucleotide sequences can be cloned out ofhybridomas, for example, by conventional hybridization techniques orpolymerase chain reaction (PCR) techniques, using synthetic nucleic acidprobes or primers whose sequences are based on sequence informationprovided herein, or known sequence information regarding genes encodingthe heavy and light chains of murine antibodies in hybridoma cells.

Techniques and protocols for engineering and production of nucleic acidsare known in the art. See, e.g., Current Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

A nucleotide sequence encoding an antibody of the invention can beoperably linked to a promoter to effect expression of the antibody in ahost cell. The sequence may include at its 5′ end a leader sequence tofacilitate expression in a host cell and/or secretion of the antibodyfrom a host cell. Suitable leader sequences are known in the art and canbe selected by the skilled person, taking account of the host cell.

In some embodiments, the nucleic acid is incorporated into a vector.Suitable vectors containing appropriate regulatory sequences, includingpromoter sequences, terminator sequences, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate, canbe obtained commercially or constructed by persons of skill in the art.For further details see, e.g., Molecular Cloning: a Laboratory Manual,2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Examples of vectors include plasmids, phages, phagemids, and cosmids, aswell as transcription and expression cassettes.

Nucleic acids encoding a HCVR and/or a LCVR disclosed herein can beincorporated (ligated) into expression vectors, which can be introducedinto host cells through conventional transfection or transformationtechniques. Accordingly, a host cell can be transformed with anexpression vector comprising a nucleotide sequence encoding a HCVRand/or a LCVR, or a fragment thereof. Examples of host cells include E.coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), and human hepatocellular carcinoma cells (e.g., HepG2).

Methods of producing an HCVR and/or LCVR, or a fragment thereof,disclosed herein are within the scope of the invention. In someembodiments, the method comprises: (a) growing a host cell containing anexpression vector encoding the HCVR and/or LCVR under conditions so thatthe host cell expresses the antibody comprising the HCVR and/or LCVR, ora fragment thereof; and (b) isolating the antibody comprising the HCVRand/or LCVR, or a fragment thereof.

Suitable conditions for antibody expression and isolation orpurification depend on the expression system employed. For example, if agene is to be expressed in E. coli, it is first cloned into anexpression vector by positioning the engineered gene downstream from asuitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signalsequence. The expressed secreted protein accumulates in refractile orinclusion bodies, and can be harvested after disruption of the cells byFrench press or sonication. The refractile bodies then are solubilized,and the proteins refolded and cleaved by methods known in the art.

If the engineered gene is to be expressed in eukaryotic host cells,e.g., CHO (Chinese hamster ovary) cells, it is first inserted into anexpression vector containing a suitable eukaryotic promoter, a secretionsignal, a poly A sequence, and a stop codon. Optionally, the vector orgene construct contains enhancers and introns. This expression vectoroptionally contains sequences encoding all or part of a constant region,enabling an entire, or a part of, a heavy or light chain to beexpressed. The gene construct can be introduced into eukaryotic hostcells using conventional techniques. The host cells express VL or VHfragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides,complete heavy or light immunoglobulin chains, or portions thereof, eachof which may be attached to a moiety having another function (e.g.,cytotoxicity).

In some embodiments, a host cell is transfected with a single vectorexpressing a polypeptide expressing an entire, or part of, a heavy chain(e.g., a heavy chain variable region) or a light chain (e.g., a lightchain variable region). In some embodiments, a host cell is transfectedwith a single vector encoding (a) a polypeptide comprising a heavy chainvariable region and a polypeptide comprising a light chain variableregion, or (b) an entire immunoglobulin heavy chain and an entireimmunoglobulin light chain. In some embodiments, a host cell isco-transfected with more than one expression vector (e.g., oneexpression vector expressing a polypeptide comprising an entire, or partof, a heavy chain or heavy chain variable region, and another expressionvector expressing a polypeptide comprising an entire, or part of, alight chain or light chain variable region).

A polypeptide comprising an immunoglobulin heavy chain variable regionor light chain variable region can be produced, for example, by growing(culturing) a host cell transfected with an expression vector encodingsuch a variable region, under conditions that permit expression of thepolypeptide. Following expression, the polypeptide can be harvested andpurified or isolated using techniques known in the art, e.g., affinitytags such as Protein A, Protein G, glutathione-S-transferase (GST), orhistidine tags.

The antibodies of the present invention can be produced by growing(culturing) a host cell transfected with, for example: (a) an expressionvector that encodes a complete or partial immunoglobulin heavy chain,and a separate expression vector that encodes a complete or partialimmunoglobulin light chain; or (b) a single expression vector thatencodes both chains (e.g., complete or partial heavy and light chains),under conditions that permit expression of both chains. The intactantibody (or antigen-binding fragment) can be harvested and purified orisolated using techniques known in the art, e.g., Protein A, Protein G,affinity tags such as glutathione-S-transferase (GST) or histidine tags.It is within ordinary skill in the art to express the heavy chain andthe light chain from a single expression vector or from two separateexpression vectors.

In some embodiments, anti-PD-1 antibodies are linked to a differentfunctional molecule or moiety, e.g., a peptide, protein, toxin,radioisotope, or cytostatic agent, for various purposes such as in vivodiagnostic imaging or a diagnostic assay. The antibodies can be linkedby chemical cross-linking or by recombinant methods. The antibodies alsocan be linked to any of various nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; or 4,179,337. The antibodies can be chemicallymodified by covalent conjugation to a polymer, for example, to increasetheir circulating half-life. Examples of polymers and methods to attachthem are described in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285;and 4,609,546.

Pharmaceutical Formulations

In some embodiments, the antibodies are formulated into pharmaceuticalcompositions suitable for administration to a mammal, e.g., a humanpatient. The compositions typically comprise one or more antibodies ofthe present invention and a pharmaceutically acceptable excipient. Theterm “pharmaceutically acceptable excipient” includes suitable solvents,dispersion media, coatings, antibacterial agents and antifungal agents,isotonic agents, and absorption delaying agents, and the like, that arecompatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is known in the art. Thecompositions also can contain other active compounds providingsupplemental, additional, or enhanced therapeutic functions. Thepharmaceutical compositions also can be included in a container, pack,or dispenser together with instructions for administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known in the art. The administrationmay be, for example, intravenous, intraperitoneal, intramuscular,intracavity, subcutaneous, intradermal, topical, inhalation,transmucosal, rectal or transdermal.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerin, propylene glycol, or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as EDTA; buffers such as acetates, citrates orphosphates; and agents for the adjustment of tonicity such as sodiumchloride or dextrose. The pH can be adjusted with acids or bases, asnecessary. Such preparations may be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. Sterilization can be accomplished, for example, byfiltration through sterile filtration membranes. For intravenousadministration, suitable carriers include, for example, physiologicalsaline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). Preferably, the pharmaceuticalcomposition is stable under the conditions of manufacture and storageand is preserved against contamination by microorganisms such asbacteria and fungi. Avoidance of microorganisms can be achieved byinclusion of antibacterial and/or antifungal agents. Examples include:parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, and sodiumchloride in the composition. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol such as glycerol,propylene glycol, liquid polyetheylene glycol, and the like, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and/or by the useof surfactants. Prolonged absorption of the injectable compositions canbe achieved by including in the composition an agent that delaysabsorption, e.g., aluminum monostearate or gelatin.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the antibodies can be combined withexcipients and used in the form of tablets, troches, or capsules.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated can be used in the formulation. Suchpenetrants are known in the art, and include, for example, detergents,bile salts, and fusidic acid derivatives. Transmucosal administrationmay be accomplished, for example, through the use of lozenges, nasalsprays, inhalers, or suppositories. For example, in case of antibodiesthat comprise the Fc portion, compositions may be capable oftransmission across mucous membranes in intestine, mouth, or lungs(e.g., via the FcRn receptor-mediated pathway as described in U.S. Pat.No. 6,030,613). For transdermal administration, the active compounds maybe formulated into ointments, salves, gels, or creams as generally knownin the art. For administration by inhalation, the antibodies may bedelivered in the form of an aerosol spray from pressured container ordispenser, which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

In some embodiments, the presently disclosed antibodies are formulatedwith carriers that protect the antibody against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used. Exemplary polymers include ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. Liposomal suspensions containingthe presently disclosed antibodies can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known inthe art. See, e.g., U.S. Pat. No. 4,522,811.

In some embodiments, pharmaceutical compositions contain, in addition toan antibody of the invention, a cytotoxic agent, cytostatic agent,anti-angiogenic agent, a tumor targeted agent, an immune stimulatingagent or immune modulating agent, or an antibody conjugated to acytotoxic, cytostatic, or otherwise toxic agent. The pharmaceuticalcomposition optionally can be employed with other therapeutic modalitiessuch as surgery, chemotherapy, and radiation.

Toxicity and therapeutic efficacy of the composition of the inventioncan be determined by conventional pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compositions that exhibit largetherapeutic indices are preferred.

A therapeutically effective dose of a therapeutic antibody can beestimated initially, e.g., from cell culture assays. Examples ofsuitable bioassays include DNA replication assays, cytokine releaseassays, transcription-based assays, PD-1/PD-L1 binding assays, creatinekinase assays, assays based on the differentiation of pre-adipocytes,assays based on glucose uptake in adipocytes, immunological assays otherassays as, for example, described in the Examples. The data obtainedfrom the cell culture assays and animal studies can be used informulating a range of dosage for use in humans. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe antibody that achieves a half-maximal inhibition of symptoms).Circulating levels in plasma may be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by a suitable bioassay. The dosage lies preferablywithin a range of circulating concentrations with little or no toxicity.The dosage may vary depending upon the dosage form employed and theroute of administration.

Generally, a therapeutically effective amount of an antibody or acomposition described herein is in the range of 0.1 mg/kg to 100 mg/kg,preferably 0.1 mg/kg to 50 mg/kg. The amount administered will depend onvariables such as the type and extent of disease or indication to betreated, the overall health of the patient, the in vivo potency of theantibody, the pharmaceutical formulation, the serum half-life of theantibody, and the route of administration.

Administration frequency can vary, depending on factors such as route ofadministration, dosage amount, serum half-life of the antibody or fusionprotein, and the disease being treated.

Therapeutic Uses

The invention provides methods of treating PD-1-mediated diseases ordisorders in a mammal, e.g., a human patient, comprising administeringan effective amount of an antibody of the present invention to a mammalin need thereof. In some embodiments, the method is a method of treatingcancer. In some embodiments, the method is a method of treatinginflammation. In some embodiments, the method is a method of treating anautoimmune disease, e.g., Crohn's disease.

As used herein, “treat”, “treating” or “treatment” means inhibiting orrelieving a disease or disorder. For example, treatment can include apostponement of development of the symptoms associated with a disease ordisorder, and/or a reduction in the severity of such symptoms that will,or are expected, to develop with said disease. The terms includeameliorating existing symptoms, preventing additional symptoms, andameliorating or preventing the underlying causes of such symptoms. Thus,the terms denote that a beneficial result is being conferred on at leastsome of the mammals, e.g., human patients, being treated. Many medicaltreatments are effective for some, but not all, patients that undergothe treatment.

As used herein, the term “effective amount” means an amount of ananti-PD-1 antibody, that when administered alone or in combination withan additional therapeutic agent to a cell, tissue, or subject, iseffective to achieve the desired therapeutic or prophylactic effectunder the conditions of administration. For example, an effective amountis one that would be sufficient to enhance or diminish the immuneresponse to bring about effectiveness of a therapy. The effectiveness ofa therapy (e.g., activation of a suppressed or deficient immuneresponse, increased cytolytic activity of T cells, increased T celleffector function, alteration of PD-1 activity associated with thenegative regulation of T-cell mediated immune response, or reduction intumor growth) can be determined by suitable methods known in the art.

When used to treat cancer, antibodies of the invention can be used aloneor in combination with another therapeutic agent. Examples of othertherapeutic agents include other checkpoint inhibitors, immunogenicagents, attenuated cancerous cells, tumor antigens (e.g., recombinantproteins, peptides, and carbohydrate molecules), antigen presentingcells such as dendritic cells pulsed with tumor-derived antigen ornucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF),and cells transfected with a gene encoding an immune stimulatingcytokine (e.g., GM-CSF); chemotherapy, radiotherapy, and surgery.

In some embodiments, an antibody of the invention is administered to acancer patient in combination with another checkpoint inhibitor. Theother checkpoint inhibitor can be targeted against PD-1 or against adifferent checkpoint molecule, e.g., TIM3, CEACAM1, TIGIT, LAG3 orVISTA. The other checkpoint inhibitor can be a small molecule or amonoclonal antibody. When the other checkpoint inhibitor is a secondPD-1 inhibitor, preferably, the mechanism of action of the second PD-1inhibitor differs from the mechanism of action of the first PD-1inhibitor. For example, the two PD-1 inhibitors can be two anti-PD-1monoclonal antibodies that bind to different epitopes on the PD-1molecule.

When used to treat cancer, antibodies of the invention can be used aloneor in combination with other checkpoint inhibitors, anti-neoplasticagents or immunogenic agents. Examples include attenuated cancerouscells, tumor antigens (including, e.g., recombinant proteins, peptides,and carbohydrate molecules), antigen presenting cells such as dendriticcells pulsed with tumor-derived antigen or nucleic acids, immunestimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), and cells transfectedwith genes encoding immune stimulating cytokines (e.g., GM-CSF; cancertreatments such as chemotherapy, radiotherapy, and surgery).

In treating certain diseases or disorders, it is desirable to diminishor suppress a patient's immune response, at least in certain tissues ofthe body. Such diseases and disorders include allergies and variousautoimmune diseases. Examples of autoimmune diseases include rheumatoidarthritis, type I diabetes mellitus, multiple sclerosis, inflammatorybowel disease, Crohn's disease, and systemic lupus erythematosis,Hashimoto's thyroiditis, ankylosing spondylitis, and graft-versus-hostdisease (GVHD). It is also desirable to suppress a patient's immuneresponse to avoid transplant rejection following tissue, skin or organtransplant.

In some embodiments, anti-PD-1 antibodies of the invention areadministered with one or more additional therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) oradministered separately. In some embodiments, the additional therapeuticagent is an immunomodulatory agent or an anti-cancer agent (e.g., achemotherapeutic agent). In separate administration, the antibody can beadministered before, after or concurrently with the agent or can beco-administered with other known therapies. Combination therapies areknown in the art. See, e.g., Hardman, et al. (eds.) (2001) Goodman andGilman's The Pharmacological Basis of Therapeutics, 10^(th) ed.,McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001)Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Philadelphia, Pa.

In some embodiments, an antibody disclosed herein is used as a targetingagent for delivery of a payload, e.g., a toxin, to a cell expressingPD-1. The method includes administering an anti-PD-1 antibody conjugatedto a payload moiety. Suitable conjugation methods are known in the art.

Non-Therapeutic Uses

In some embodiments, antibodies of the invention are used fornon-therapeutic purposes, such as diagnostic tests and assays. Forexample, the invention provides a method of diagnosing a PD-1-mediatedadaptive immune resistance in a patient who has cancer. The methodcomprises contacting a tumor microenvironment in the patient with anantibody disclosed herein that has been labeled with a detectablemoiety; and detecting expression of PD-1 on immune cells, e.g., CD8+ Tcells; B cells; and macrophages, within the tumor microenvironment.

Adaptive immune resistance includes suppression of a host immuneresponse as a result of activation of a PD-1 signaling pathway in immunecells of the host. For example, cancer tissue suppresses a host immuneresponse by upregulation of PD-L1 and its binding to PD-1 on immunecells on T cells (such as CD8+ T cells); B cells; and macrophages.

A diagnostic method utilizing an antibody of the invention to detectPD-1 expression also can comprise an agent for detecting expression ofPD-L1 on immune cells within the tumor microenvironment. Such adiagnostic method can be performed in vivo, or on a biopsy sample from apatient, wherein the tumor microenvironment is present in a tumorbiopsy.

Modifications of antibodies for diagnostic purposes are well known inthe art. For example, antibodies may be modified with a ligand groupsuch as biotin, or a detectable marker group such as a fluorescentgroup, a radioisotope, or an enzyme. Antibodies of the invention can belabeled using conventional techniques. Suitable detectable labelsinclude: fluorophores, chromophores, radioactive atoms, electron-densereagents, enzymes, and ligands having specific binding partners. Enzymestypically are detected by their reaction products. For example,horseradish peroxidase can be detected through conversion oftetramethylbenzidine (TMB) to a blue pigment, quantifiable with aspectrophotometer. For detection, suitable binding partners includebiotin and avidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. Other permutations andpossibilities will be readily apparent to those of ordinary skill in theart.

Antibodies of the invention also can be used to detect the presence ofPD-1 in biological samples. The amount of PD-1 detected can becorrelated with the expression level of PD-1, which, in turn, iscorrelated with the activation status of immune cells, e.g., activated Tcells, B cells, and monocytes, in the subject.

Detection methods that employ antibodies are known in the art, andinclude ELISA, radioimmunoassay, immunoblot, Western blot,immunofluorescence, and immunoprecipitation techniques. Antibodies ofthe invention can be provided in a diagnostic kit that incorporates oneor more of these techniques to detect PD-1. Such a kit may contain othercomponents, packaging, instructions, or material to aid in the detectionof PD-1 protein.

EXAMPLES

The following Examples are merely illustrative, and are not intended tolimit the scope or content of the invention in any way.

Example 1 Identification of PD-1 Antibodies

A. Immunization of Mice with PD-1

Balb/C, C57BL/6 or NZW/B female mice aged 4-8 weeks were immunized in astandard prime/boost regimen employing standard adjuvant mixtures. Asoluble extracellular domain of human PD-1 (AA1-167) expressed with aC-terminal polyhistidine sequence (SinoBiological #10377-H08H) was usedfor immunizations. Cohorts of mice were primed with 50 μg of recombinantPD-1 and (1) complete Freund's adjuvant (Sigma-Aldrich #263810) or (2)alhydrogel (Invivogen). Two to three weeks later, animals in each groupwere boosted with 50 μg of soluble PD-1 with (1) incomplete Freund'sadjuvant (Sigma-Aldrich #263910) or (2) alhydrogel. Serum titres werecollected after each antigen boost and assayed by ELISA for reactivityand antibody isotype class switching. The same protein used forimmunizations was immobilized onto 96 well assay plates (Nunc MAXISORP)at a concentration of 1 μg/mL. Serial dilutions of the sera fromimmunized animals were then tested for binding to PD-1.

B. Screening of Antibodies for Binding to Human PD-1

Cells, fresh or thawed from cryopreserved samples, from bone marrow,lymph nodes or the spleen were carried in standard tissue culture medium(LifeTech RPMI with 10% low IgG). Cells were interrogated forantigen-specific B cells, unstimulated or stimulated, using LPS(Invivogen) at a concentration of 20 ng/mL. Cells, unstimulated orstimulated, were then loaded at a stochastic cellular density to favorsingle cell per well loading onto microwell arrays (MWAs) as describedin U.S. Pat. Nos. 7,776,553 and 8,772,049.

A functionalized capture surface, coated with two mixtures of polyclonalanti-mouse IgG antibodies (Jackson Immunoresearch #715-005-150,#115-005-146), was then used to hermetically seal the ordered microwellarray. After two hours, the capture surface(s) was removed from themicrodevices and processed as described previously (Ogunnyi et al.Nature Protocols, 2009). The microarray capture surface, representing amirror image of the cells in the microwell array, contained the secretedoutput of the B cells. The antibodies secreted by the B cells innanowells and captured were then assayed for reactivity against humanPD-1, or an unrelated antigen, and also assayed for IgG versus IgMreactivity (JacksonImmunoresearch #115-005-044, #115-005-164).

After the protein microarrays were scanned, putative antibody cloneswith the desired specificity and antibody isotype class werebioinformatically identified by standard data quality metrics. Accordingto methods previously described (Ogunnyi et al., Vaccine 2014),microarray images were analyzed using Molecular Devices GenePixsoftware. Microarray features were analyzed for false positivity,co-variance, and signal-to-noise ratios. Features with the correctattributes, e.g., specific for PD-1 and IgG, were then nominated forcellular retrieval. This automated pick-list was then generated from 4to 12 microdevices, and cells that had secreted antibodies with desiredcharacteristics were isolated from the microdevices and placed intostandard SBS microtitre assay plates for further processing.

C. Isolation of Antibodies that Bind PD-1

Single antibody-producing cells identified from screening were used forsingle cell molecular biology in order to isolate the genetic sequencesencoding antibody heavy and light chains (Tiller et al., J. Immunol.Methods 350:183-93 (2009)). The genes encoding the specific antibodiesthat recognize human PD-1 were retrieved using single cell RT-PCR.Retrieved cells were placed into reverse transcription buffer, and themRNA from each individual cell was reverse transcribed (LifeTechSuperScript III) into cDNA. After generating these amplicons by standardnested polymerase chain reaction(s) (PCR), these amplicons weresubjected to direct sequencing. After analysis by Phred software using aPhred 0.05 cut-off value, sequences were sub-cloned into PCR 2.1(LifeTech) or other standard vector backbones for further propagation.Phred is software that reads DNA sequencing trace files, calls bases,and assigns a quality value to each called base. See, e.g., Ewing andGreen, Genome Research 8:186-94 (1998).

These DNA sequences were then bioinformatically filtered for sequencequality and organized into a sequence database for cladistics analysis(distance or parsimony), in order to identify how many unique antibodyclades were isolated that recognize PD-1. These analyses identifiedapproximately 20 unique clades (groups) of sequences that recognizehuman PD-1.

D. Antibody Construction

Complete antibody variable regions comprising a pair of heavy and lightchain variable regions (Tables 1 and 2) were reformatted into plasmidswith the proper elements for transient ectopic expression in mammaliancell lines, e.g., HEK293 or CHO, using standard molecular biologytechniques. For example, the variable heavy (VH) and variable light (VL)cDNA sequences were sub-cloned into the vector backbone pFUSE-CHIg-mG1(InVivoGen), which contains an IL2 signal sequence, as well as anin-frame murine Fc-domain. A sequence verified consensus sequence foreach antibody VH and VL gene was engineered, using PCR primers, withrestriction sites. The expression vector of choice and the PCR ampliconswere then digested with restriction enzymes and then ligated togetherfor transformation of E. Coli. Resulting expression clones were sequenceverified.

The sequences of the individual heavy chain and light chain variableregions are shown in FIG. 1 (HCVR) and FIG. 2 (LCVR). Thecomplementarity determining regions (CDRs) and framework regions (FRs)are indicated. Tables 1 and 2 list each HCVR or LCVR by clone name andcorresponding sequence identifier. The corresponding sequences are shownin FIGS. 1 and 2.

TABLE 1 Heavy Chain Variable Region Sequence Designations andIdentifiers Heavy Chain Variable Region SEQ ID NO: 100244_C8VH3 1100258_F6VH4 2 100246_F7VH3 3 100244_C7VH10 4 100258_C1VH4 5100392_C4VH5 6 100394_D5VH9 7 100394_G1VH1 8 100392_C5VH6 9 100392_H3VH610 100394_F1VH8 11 100388_C1VH2 12 100388_C1VH6 13 100388_G1VH5 14100392_B4VH10 15 100392_H4VH9 16 100392_D2VH7 17 100388_D4VH3 18100392_A6VH8 19 100246_A10VH1 20 100411_C2VH3 21 100413_D2VH9 22100388_H4VH1 23 100392_C3VH7 24 100413_E1VH9 25 100413_H1VH3 26

TABLE 2 Light Chain Variable Region Sequence Designations andIdentifiers Light Chain Variable Region SEQ ID NO: 100245_C8VK5ml 27100245_C8VK5 28 100259_F6VK1m1 29 100259_F6VK1 30 100247_F7VK2 31100389_C1VK2 32 100395_F1VK4 33 100393_C4VK8 34 100395_G1VK4 35100389_G1VK1 36 100393_C5VK10 37 100393_C5VK7 38 100389_C1VK1 39100393_D2VK6 40 100395_D5VK7 41 100393_H3VK2 42 100393_B4VK10/H4VK6 43100259_C1VK1 44 100414_E1VK5 45 100414_H1VK6 46 100389_D4VK5 47100393_A6VK5 48 100389_H4VK4 49 100393_C3VK10 50 100414_D2VK6 51100412_C2VK7 52 100247_A10VK3 53

Example 2 Antibody Characterization

A. Hit Confirmation and Specificity

Supernatants from transiently transfected mammalian cell lines were usedto test for immunoglobulin (Ig) expression, antigen specificity, andantigen affinity. These assays were ELISA-based, using reagents thatrecognize IgG, as well as the soluble ECD of PD-1 as an Fc-fusionprotein (SinoBiological “CD279-Fc”). While the screening immunogen wasthe soluble form of PD-1, the form used for binding confirmation was afusion protein comprising the ECD of human PD-1 with a human Fc domain.Other immune checkpoint proteins in the same biochemical configurationswere used as specificity controls, e.g., CD28, GITR. Proteins wereimmobilized in wells of a 96 well assay plate (Nunc MAXISORB) andsupernatants from transfected HEK293 cells were used to assess bindingof reformatted anti-PD-1 antibodies, produced as described above. Theseexperiments enabled determination of binding specificity and affinity.

FIG. 3 shows an example of an ELISA-based binding assay. Various VH andVK were paired to yield 32 clones (clone pairings shown in box of FIG.3), which were expressed in HEK293 cells and assayed for binding toPD-1-HIS(tag). The results represent an average of two experiments.

B. Cellular Binding

Recombinant antibodies from cultured supernatants were also used incell-based binding studies. HEK293 cells were transfected (Qiagen,SuperFect) with expression plasmids encoding the Ig heavy and lightchains of anti-PD-1 antibodies. After 3-5 days, recombinant antibodiesin the supernatants of transfected cells were harvested. Stable HEK293cells expressing human PD-1 or primary cells were used for thecell-based binding confirmation studies. Antibodies from culturedsupernatants were assessed for binding to cell surface PD-1 usingfluorescently labeled anti-mouse IgG (polyclonal) antibody (JacksonImmunoresearch).

Fluorescence microscopy studies revealed that a number of anti-PD-1antibodies obtained from transfection supernatants bound to PD-1expressed on the surface of HEK293 cells. Binding of four mouseanti-PD-1 antibodies including, 100244_C7VH10_100245_C8VK5m1 (comprisingthe HCVR corresponding to SEQ ID NO: 4 and LCVR corresponding to SEQ IDNO: 27), were detected with anti-mouse κ-PE (secondary antibody).Similarly, in a separate study, binding of five mouse anti-PD-1antibodies including, 100392_C5VH6_100393_C5VK7, comprising the HCVRcorresponding to SEQ ID NO: 9 and LCVR corresponding to SEQ ID NO: 38,were detected with anti-mouse IgG1-AF488 (secondary antibody). In bothstudies, commercially available mouse anti-PD-1 antibody and isotypecontrol mouse IgG1 anti-PD-1 were used as controls. Table 3 summarizesresults of tests of binding of various anti-PD-1 antibodies to PD-1expressed on the surface of HEK293 cells. Strong binding is denoted as“+++”, medium binding is denoted as “++”, and weak binding is denoted as“+”.

TABLE 3 Summary of PD-1 mAb binding to cell-surface expressed PD-1Binding to Human PD-1 Expressed on mAb Mouse anti-human PD-1 Abs HEK293Surface Name 100244_C7VH10_100245_C8VK5 ++ 244C7100244_C7VH10_100245_C8VK5m1 ++ 244C7m1 100244_C8VH3_100245_C8VK5 ++244C8 100244_C8VH3_100245_C8VK5m1 ++ 244C8m1 100246_F7VH3_100247_F7VK2++ 246F7 100258_C1VH4_100259_C1VK1 + 258C1 100258_F6VH4_100259_F6VK1 ++258F6 100258_F6VH4_100259_F6VK1m1 ++ 258F6m 100392_C4VH5_100393_C4VK8+++ 392C4 100394_D5VH9_100395_D5VK7 ++ 394D5 100394_G1VH1_100395_G1VK4++ 394G1 100388_C1VH2_100389_C1VK1 ++ 388C12A 100388_C1VH2_100389_C1VK2++ 388C12B 100388_C1VH6_100389_C1VK1 ++ 388C16A100388_C1VH6_100389_C1VK2 ++ 388C16B 100388_G1VH5_100389_G1VK1 − 388G1100392_B4VH10_100393_B4VK10 − 392B4 100392_C5VH6_100393_C5VK7 ++ 392C5A100392_C5VH6_100393_C5VK10 ++ 392C5B 100392_D2VH7_100393_D2VK6 ++ 392D2100392_H3VH6_100393_H3VK2 − 392H3 100392_H4VH9_100393_H4VK6 + 392H4100394_F1VH8_100395_F1VK4 − 394F1 100246_A10VH1_100247_A10VK3 +++ 246A10100388_D4VH3_100389_D4VK5 +++ 388D4 100388_H4VH1_100389_H4VK4 − 388H4100392_A6VH8_100393_A6VK5 ++ 392A6 100392_C3VH7_100393_C3VK10 − 392C3100411_C2VH3_100412_C2VK7 ++ 411C2 100413_D2VH9_100414_D2VK6 ++ 413D2100413_E1VH9_100414_E1VK5 ++ 413E1 100413_H1VH3_100414_H1VK6 − 413H1mouse anti-PD-1 cl. EH12.2H7 (1 μg/mL) ++ n/a isotype control mouse IgG1(1 μg/mL) − n/a

C. Cell-Based Activity

Antibodies that recognize PD-1 with high affinity were selected for usein cell-based assays to test for agonist and antagonist activity. Usingstandard ex vivo activation conditions with primary human cells,selected anti-PD-1 antibodies were used for cell-based assays inmicrowell array devices, using conditions that have been previouslycharacterized (see, e.g., Varadarajan et al., 2012, Proc. Nat'l Acad.Sci. 109:3885-3890) to assess effects of these antibodies on effectorcell function.

Peripheral blood mononuclear cells (PBMCs) obtained from de-identifieddonors via a commercial source were used in these studies. PBMCs wereplaced into wells of a 96-well assay plate previously coated with platebound anti-CD3 (OKT3) at a concentration of 1 μg/mL. In addition toTCR-mediated stimulation, cells were treated with PBS (control), solublerecombinant human PD-L1 (shPD-L1) (SinoBiological) at 20 μg/mL, orshPD-L1 with anti-PD-1 antibody (10 μg/mL). Cellular proliferation wasassayed by ELISA for IL-2 (R&D Systems, #D2050) in the supernatant ofcell cultures, or by direct measurement of secreted IFNγ at the singlecell level (Varadarajan, supra) using microwell array devices.

FIG. 4 summarizes results from assays to measure the effectiveness ofanti-PD-1 antibodies in relieving PD-L1 dependent inhibition ofactivation of human peripheral blood mononuclear cells (PBMCs).Treatments of cells were carried out in 96-well plates for 3-5 days. Alltreatments included plate-bound CD3 and soluble CD28 in conventionalmedia. Cells were treated additionally with nothing (positive control);with PD-L1 alone; or with PD-L1 plus an anti-PD-1 antibody (EH12.2H7(BioLegend Products, San Diego, Calif.), j 105 (eBioscience, San Diego,Calif.), pembrolizumab, 246A10 or 244C8). At the end of the treatmentperiod, cells were transferred to a microwell array device for IFNγdetermination at the level of individual cells, with sample cellpopulations in the range of approximately 50-100 cells. The resultsshown in FIG. 4 illustrate antagonism of PD-1 activity by anti-PD-1antibodies. These anti-PD-1 antibodies blocked the inhibitory effect ofPD-L1, thus decreasing PD-1/PD-L1 (or PD-1/PD-L2) mediated inhibition ofcellular immune response.

D. Differential T Cell Activation

In a first type of cell-based activation assay, commercially-sourcedhuman PBMCs (peripheral blood mononuclear cells) (Research BloodComponents, Allston, Mass.) were analyzed by flow cytometry to test fordifferential T cell activation in response to PD-1 blockade by differentanti-PD-1 antibodies in vitro. CD4 and CD8 were used as T cell markers.The relative extent of T cell activation was inferred from measuringproduction of the effector cytokines, interferon gamma (IFNγ) and tumornecrosis factor-alpha (TNFα). The experiments were conducted essentiallyas follows. Approximately 500,000 to 750,000 PBMCs were incubated forthree days in the presence of 1 μg/mL anti-CD3 (clone HIT3a), 50 ng/mLanti-CD28 (clone CD28.2) and 20 μg/mL anti-PD-1 antibody or isotypecontrol. At the end of the 3-day incubation period, cells were treatedwith Brefeldin A for 6 hours and then subjected to extracellularstaining for CD4, CD8, CD69, CD25, PD-L1, or other extracellular markersconjugated to fluorophores. Cells were then fixed, permeabilized, andstained for intracellular markers including IFNγ (antibody clone 4S.B3).Data were collected by flow cytometry using a FACSCALIBUR™ flowcytometer (Becton Dickinson, Franklin Lakes, N.J.), and analyzed usingFLOWJO™ software (FlowJo, LLC, Ashland, Oreg.). The antibodies testedwere pembrolizumab, clone EH12.2H7 (BioLegend), and anti-PD-1 antibodies388D4, 413E1, 246A10 and 244C8 of the present invention. Underconditions of suboptimal activation (achieved by the treatment withanti-CD3 and anti-CD28), which may mimic activation conditions thatoccur in vivo, antibodies 388D4, 413E1, 246A10 and 244C8 in these testselicited similar secretion levels, or enhanced secretion levels of IFNγand TNFα, as compared to EH12.2H7 or pembrolizumab (Table 4). The datain Table 4 are compared graphically in FIG. 5.

TABLE 4 Summary of differential T cell activation in response to PD-1blockade by different anti-PD-1 antibodies CD8 IFNγ CD4 IFNγ CD8 TNFα %positive % positive % positive Negative 4.6 1.64 3.73 control CD3/CD2818.2 6.31 6.19 pembrolizumab 28.4 14.1 13 388D4 31.2 21.2 8.56 413E1 2720.2 11.1 246A10 26.2 20.3 14.9 244C8 19.9 9.64 14.5 EH12.2H7 19.5 12.814.1 Positive 73.7 61.5 37.3 control

In a second set of experiments, human PBMCs were tested for reactivityin antigen recall assays using CMV (cytomegalovirus) (IMMUNOSPOT®,Shaker Heights, Ohio). PBMCs were purified from whole blood of healthydonors (Research Blood Components, Allston, Mass.). The PBMCs wereincubated with ready-to-use peptide antigen solutions (AstarteBiologics, Bothell, Wash.) without or with anti-PD-1 antibodies. Twocommercial stage anti-PD-1 antibodies, pembrolizumab and nivolumab, wereused as benchmarks for this experiment. Compared to antibody isotypecontrol(s), anti-PD-1 antibodies induce increased levels of IFNγ, a keyeffector cytokine in T cell antigen recall biology (FIG. 6).

In a third set of experiments, anti-PD-1 antibodies were used with PBMCsin MLR (mixed lymphocyte reaction) assays. In these assays, secretion ofIL-2 or IFNγ was the experimental cytokine readout (FIG. 7A). Activationmarkers such as CD25 were also evaluated (FIG. 7B). Results aresummarized in FIGS. 7A and 7B. In this assay, clone 388D4 appears toinduce cytokine release and CD25 upregulation similar to nivolumab. Inmultiple assays, clone 244C8 appears to induce increased levels ofcytokine release (IFNγ) compared with 388D4 or nivolumab. T cellsincubated with 244C8 also appear to exhibit a higher degree ofactivation as inferred from CD25 expression (FIG. 7B).

These data indicate that some of the anti-PD-1 antibodies of the presentinvention, e.g., 388D4, induce increased cytokine release in a mannersimilar to pembrolizumab and nivolumab, while other antibodies, e.g.,244C8, elicit physiological responses that are measurably different fromthe responses elicited by pembrolizumab and nivolumab.

E. Peptide-Based Epitope Mapping

Synthetic overlapping peptides based on the human PD-1 sequence(15-mers) (Sigma-Aldrich PEPscreen, Saint Louis, Mo.) were used inepitope mapping experiments. The peptides used are listed in Table 5below.

TABLE 5 Human PD-1 peptide sequences used for epitope mapping PeptideN-term C-term Name mod Sequence Mod SEQ ID NO: PD101 [H] MQIPQAPWPVVWAVL[OH] 54 PD102 [H] APWPVVWAVLQLGWR [OH] 55 PD103 [H] VWAVLQLGWRPGWFL [OH]56 PD104 [H] QLGWRPGWFLDSPDR [OH] 57 PD105 [H] PGWFLDSPDRPWNPP [OH] 58PD106 [H] DSPDRPWNPPTFSPA [OH] 59 PD107 [H] PWNPPTFSPALLVVT [OH] 60PD108 [H] TFSPALLVVTEGDNA [OH] 61 PD109 [H] LLVVTEGDNATFTCS [OH] 62PD110 [H] EGDNATFTCSFSNTS [OH] 63 PD111 [H] TFTCSFSNTSESFVL [OH] 64PD112 [H] FSNTSESFVLNWYRM [OH] 65 PD113 [H] ESFVLNWYRMSPSNQ [OH] 66PD114 [H] NWYRMSPSNQTDKLA [OH] 67 PD115 [H] SPSNQTDKLAAFPED [OH] 68PD116 [H] TDKLAAFPEDRSQPG [OH] 69 PD117 [H] AFPEDRSQPGQDCRF [OH] 70PD118 [H] RSQPGQDCRFRVTQL [OH] 71 PD119 [H] QDCRFRVTQLPNGRD [OH] 72PD120 [H] RVTQLPNGRDFHMSV [OH] 73 PD121 [H] PNGRDFHMSVVRARR [OH] 74PD122 [H] FHMSVVRARRNDSGT [OH] 75 PD123 [H] VRARRNDSGTYLCGA [OH] 76PD124 [H] NDSGTYLCGAISLAP [OH] 77 PD125 [H] YLCGAISLAPKAQIK [OH] 78PD126 [H] ISLAPKAQIKESLRA [OH] 79 PD127 [H] KAQIKESLRAELRVT [OH] 80PD128 [H] ESLRAELRVTERRAE [OH] 81 PD129 [H] ELRVTERRAEVPTAH [OH] 82PD130 [H] ERRAEVPTAHPSPSP [OH] 83 PD131 [H] VPTAHPSPSPRPAGQF [OH] 84

Each peptide was incubated with each antibody for one hour, to allowpeptide-antibody complex formation. Then each of these antibody-peptidemixtures was used in a conventional ELISA, in which human PD-1 wasimmobilized on 96-well plates. ELISA plates were coated with 100 ng/wellof PD-1-His Tag (Sino Biological, North Wales, Pa.; #10377-H08H-50) incarbonate buffer, pH 9.6. After washing, plates were blocked with 4%milk PBS, 0.05% Tween-PBS (blocking buffer). After blocking and washingof the plates, the peptide-antibody mixtures were incubated with theimmobilized human PD-1. After washing, the plates were developed byincubation for 1 hour with goat HRP-conjugated anti-mouse IgG (JacksonImmunoResearch, West Grove, Pa.; #115-035-071) and addition of 100 μl ofTMB solution (ThermoScientific, Waltham, Mass.; #PI-34022). Opticaldensities were measured at the appropriate wavelength, using an ELISAmicroplate reader. This enabled quantitative assessment of whichpeptides complexed with the antibody and then inhibited antibody bindingto human PD-1. FIG. 8 summarizes the binding results of five antibodyclones (246A10, 244C8, 388D4, 413D2, and 413E1).

F. Biophysical Characterization of Anti-PD-1 Antibodies

Biophysical characteristics of certain anti-PD-1 antibodies wereanalyzed by biolayer interferometry (BLI), using the ForteBio Octet Redsystem (Pall Corporation, Menlo Park, Calif.). The antibodies wereimmobilized on BLI biosensors and then incubated with the solubleextracellular domain of human PD-1. Using standard biophysics methods,apparent on-rates and off-rates were then inferred for PD-1 withanti-PD-1 antibodies. These values were used to generate apparentaffinity values (K_(D) values), which are listed in Table 6.

TABLE 6 IgG K_(D) (nM) k_(on) (1/Ms) k_(dis) (1/s) R_(max) Full X² FullR² 246A10 76.4 1.61 × 10⁵ 1.23 × 10⁻² 0.10 0.01 0.99 244C8 15.1 2.13 ×10⁵ 3.22 × 10⁻³ 0.12 0.01 1.00 413D2 8.20 2.75 × 10⁵ 2.26 × 10⁻³ 0.150.02 0.99 393C5 2.44 4.75 × 10⁵ 1.16 × 10⁻³ 0.15 0.01 1.00 388D4 2.693.71 × 10⁵ 9.99 × 10⁻⁴ 0.16 0.01 1.00 413E1 58.9 5.07 × 10⁵ 2.99 × 10⁻²0.12 0.01 0.99

G. Selectivity of Anti-PD-1 Antibodies

To assess selectivity of the anti-PD-1 antibodies, ELISA was used togenerate dose response curves for binding of the anti-PD-1 antibodies toseveral immunomodulatory cell surface proteins. Recombinant solubleextracellular domains (ECDs) of ICOS (inducible T-cell costimulator),PD-1, CD28 or CTLA4 (R&D Systems, Minneapolis, Minn.) were coated ontoELISA assay plates. Binding of each anti-PD-1 antibody and each controlantibody to each target protein was then assessed over a range ofantibody concentrations (FIGS. 9A-9D).

These experiments demonstrated that anti-PD-1 antibodies 388D4, 413E1,244C8 and 246A10 bind to the PD-1 ECD with high specificity, showing nobinding to three structurally related Ig-superfamily protein members

Example 3 Humanization of Anti-PD-1 Antibodies

Humanization of selected anti-PD-1 antibodies was performed in order toreduce the apparent immunogenicity of the mouse-based antibodies. Usingantibody engineering information well known in the art, and conventionalbioinformatics tools, amino acid sequences of certain murine anti-PD-1antibodies of the invention were analyzed and compared against knownhuman antibody sequences. Based on these analyses and comparisons,certain human sequences were chosen for conventional murine CDRgrafting, and inclusion of suitable back mutations. In tests for bindingto human PD-1, these humanized antibodies were evaluated with respect tocriteria such as affinity, avidity, binding kinetics, and biochemicalbehavior such as aggregation as well as expression levels. The HCVR andLCVR amino acid sequences of certain humanized antibodies displayingdesirable characteristics (e.g., binding to PD-1) are shown in Tables 7and 8, respectively. FIGS. 10A and 10B show amino acid sequencealignments of the humanized heavy or light chain variable regionsequences with the indicated murine heavy (100388_D4_VH3 or100244_C8_VH3) or light chain (100389_D4_VK5 or 100245_C8_Vk5m1)variable region sequences.

TABLE 7 Humanized heavy chain variable region sequences SEQ HCVR IDdesignation Amino Acid Sequence NO 100244_C8_HC1QVQLVQSGAEVKKPGASVKVSCKASGYTFT 85 SYWMHWVRQAPGQGLEWMGMIDPSNSETSLNQKFQGRVTMTVDKSTNTVYMELSSLRSE DTAVYYCARSRGNYAYEMDYWGQGTLVT VSS100244_C8_HC2 EVQLVQSGAEVKKPGASVKVSCKASGYTFT 86SYWMHWVRQAPGQGLEWMGMIDPSNSETS LNQKFQGRVTLNVDKSTNTAYMELSSLRSEDTAVYYCARSRGNYAYEMDYWGQGTLVT VSS 100244_C8_HC3EVQLVQSGTEVTKPGASVKVSCKASGYTFT 87 SYWMHWVRQAPGQGLEWLGMIDPSNSETTLNQKFQGRVTMTVDKSTNTVYMELTSLRSE DTAVYYCARSRGNYAYEMDYWGQGTLVT VSS100388_D4_HC1 EVQLVQSGAEVKKPGASVKVSCKASGYTFT 88DYEMHWVRQAPGQGLEWMGIIDPGTGGTA YNQKFQGRVTMTADKSTSTVYMELSSLRSEDTAVYYCTSEKFGSNYYFDYWGQGTLVTV SS 100388_D4_HC2EVQLVQSGAEVKKPGASVKVSCKASGYTFT 89 DYEMHWVRQAPGQGLEWMGIIDPGTGGTAYNQKFQGRVTMTADKSTNTVYMELSSLRSE DTAVYYCTSEKFGSNYYFDYWGQGTLVTV SS100388_D4_HC3 EVQLVQSGAEVKKPGASVKVSCKASGYTFT 90DYEMHWVRQAPGQRLEWMGVIDPGTGGT AYNQKFQGRVTITADKSASTAYMELSSLRSEDTAVYYCTSEKFGSNYYFDYWGQGTLVT VSS

TABLE 8 Humanized light chain variable region sequences SEQ LCVR IDdesignation Amino Acid Sequence NO 100245_C8_LC1EIVLTQSPATLSLSPGERATLSCRASSSVSSN 91 YLYWYQQKPGQAPRLLIYSTSNRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSS YPPTFGQGTKLEIK 100245_C8_LC2DIVLTQSPATLSLSPGERATLSCRASSSVSSN 92 YLYWYQQKPGQAPRLLIYSTSNLATGIPARFSGSGSGTDYTLTISSLEPEDFAVYFCHQWSS YPPTFGQGTKLEIK 100245_C8_LC3DIVLTQSPGTLSLSPGEKVTLSCRASSSVSSN 93 YLYWYQQKPGQAPRLVIYSTSNLATGIPDRFSGSGSGTDYTLTISRLEPEDFAVYFCHQWSS YPPTFGQGTKVEIK 100389_D4_LC1DVVMTQSPLSLPVTLGQPASISCRSSQTIVHS 94 DGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLTFGQGTKLEIK 100389_D4_LC2DIVMTQSPLSLPVTLGQPASISCRSSQTIVHS 95 DGNTYLEWYQQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLTFGQGTKLEIK 100389_D4_LC3DIVMTQTPLSSPVTLGQPASISCRSSQTIVHS 96 DGNTYLEWYQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYY CFQGSHVPLTFGQGTKLEIK

TABLE 9 HCVR and LCVR pairings for humanized antibodies HumanizedAntibody Light Chain Designation Heavy Chain Variable Region VariableRegion 244C8-1 100244_C8_HC1 (SEQ ID NO: 85) 100245_C8_LC1 (SEQ ID NO:91) 244C8-2 100244_C8_HC1 (SEQ ID NO: 85) 100245_C8_LC3 (SEQ ID NO: 93)244C8-3 100244_C8_HC2 (SEQ ID NO: 86) 100245_C8_LC1 (SEQ ID NO: 91)388D4-1 100388_D4_HC3 (SEQ ID NO: 90) 100389_D4_LC1 (SEQ ID NO: 94)388D4-2 100388_D4_HC3 (SEQ ID NO: 90) 100389_D4_LC3 (SEQ ID NO: 96)388D4-3 100388_D4_HC1 (SEQ ID NO: 88) 100389_D4_LC3 (SEQ ID NO: 96)

Example 4 Antibody-Ligand Competition for Binding to PD-1

A. Competitive Binding Assays

It was found that while some of the anti-PD-1 antibodies disclosedherein competitively inhibit binding of PD-1 ligands, others do not. Forexample, in surface plasmon resonance-based competitive binding assaysand flow cytometry-based competitive binding assays, it was found thatantibody 388D4 competitively inhibits binding of PD-L1 to PD-1, but doesnot inhibit binding of PD-L2. In contrast, it was found that antibody244C8 does not competitively inhibit binding of PD-L1 or PD-L2.

Competitive Binding Analysis was performed on humanized IgG₄ antibodies244C8-2 and 388D4-2, using ForteBio Biolayer Interferometry (BLI).Humanized IgG antibody was immobilized to AHC biosensors by loading 3μg/mL IgG to a target level of 1.0 nm over a 160 second load time. Asingle concentration (100 nM) of active PD-1, plus an appropriatenegative control to correct for drift, was bound the immobilized IgG. ApH of 7.4 was used for association and dissociation. The bound PD-1 wasthen exposed to seven concentrations of PD-L1 (9, 3, 1, 0.333, 0.111,0.037, and 0 μM) or PD-L2 (2000, 666.7, 222.2, 74.1, 24.7, 8.2, and 0nM). The association/dissociation of PD-L1 and PD-L2 to the mAb/PD-1complex immobilized on biosensor tips was evaluated.

Materials used in these assays were as follows: PD1 (His Tag): ABCAM,Cat# ab174035, Lot# GR199119-1, 100 mg; PD-L1 (His Tag): SinoBiological, Cat#10084-H08H, Lot# LC098E0901, 200 mg; PD-L2 (His Tag):Sino Biological, Cat#10292-H08H, Lot# LC07DE3022, 100 mg; PD1-Fc: R&DSystems, Cat#1086-PD, Lot# FVQ0413051, 50 mg; Anti-Human IgG-Fc Capture(AHC) Biosensors: ForteBio, Cat#18-5060, Lot#1501211; 1× Kinetic Buffer:20 mM Phosphate, 150 mM NaCl, 0.02% Tween-20, 0.05% Sodium Azide, 0.1mg/ml BSA, pH 7.4; Test Samples: Humanized IgG₄—244C8-2 (3.04 mg/mL),Humanized IgG₄—388D4-2 (2.89 mg/mL).

The flow cytometry assays were conducted essentially as follows. HEK293cells expressing PD-1 were incubated with 10 μg/ml of an isotypeantibody (negative control), commercially available antibody EH12.2H7(positive control), antibody 388D4, or antibody 244C8. Cells were washedand stained with soluble PD-L1-Ig protein fluorescently labeled withAlexa-488. Cells were washed again, and PD-L1 binding (by displacingpreviously bound antibody) was assessed by fluorescence activated cellsorting (FACS) analysis. Representative results are shown in FIGS.11A-11D.

Example 5 Human Cell Based Assays

A. Human Cells

Human tumor tissue procurement and tumor dissociation were as follows.Fresh tumor samples from NSCLC patients undergoing surgical resection oftumors were obtained from the Cooperative Human Tissue Network, NationalCancer Institute. Analysis was performed using single-cell suspensionsof tumor cells from these tumor samples.

Solid tumor biopsy samples were mechanically disrupted into single-cellsuspensions using a gentleMACS Dissociator (Miltenyi Biotec) withenzymes A, H and R. The single-cell suspensions were then prepared forcell counting and initial FACS analysis.

B. FACS Analysis

For FACS analysis, anti-CD45-PerCP-Cy5.5 (clone 2D1), anti-CD4-PE-Cy7(SK3), anti-CD8-FITC (SK1), anti-BTLA-Biotin (MIH26), anti-CTLA-4-PE(14D3), and anti-LAG-3-APC (3DS223H) were purchased from eBioscience.Anti-CD25-BV605 (2A3), anti-PD-1-BV605 (EH121), and Streptavidin-BV711were purchased from BD Bioscience. Anti-CD45RABV421 (HI100), anti-CCR7AlexaFluor647 (G043H7), and anti-Tim-3-BV421 (F38-2E2) were purchasedfrom Biolegend. Heterogeneous cell suspensions prepared from dissociatedprimary tumors (as described above) were washed, resuspended in PBS, andblocked with a commercial Fc blocking reagent (BD Biosciences). Viablecells were identified by lack of dead cell staining positivity, and bynegativity for EpCAM expression. CD45-positive cells were gated for CD4or CD8 expression, and then the cells were assessed for expression ofPD-1, TIM3, LAG3 or TIGIT. These FACS data are shown below, in Table 10,with results expressed as percentage of positive cells.

TABLE 10 Results of FACS analysis % % % % % % % % % EpCAM− % CD4⁺ CD8⁺CD4⁺ CD8⁺ CD4⁺ CD8⁺ CD4⁺ CD8⁺ Tumor CD45+ CD3+ PD1⁺ PD1⁺ TIM3⁺ TIM3⁺LAG3⁺ LAG3⁺ TIGIT⁺ TIGIT⁺ WD-36444 25.7 14 55 75 5.5 13 <1 <1 6 6.3WD-36571 10.3 6.3 47 64 1.8 <1% 0 0 12.6 21 WD-36686 21.6 17 55 84 6 160 0 20 15 WD-36790 16.8 10.4 38 68 1 5.2 <1 <1 33 47 WD-36904 12.8 7 6372 9.5 24.5 <1 <1 41 25.6 M115801A2 3.4 2.9 79 84 22 16 1.4 1.6 56 35WD-36923 1.6 0.9 53 51 27 24.5 WD-36988 8.9 7 58 93 22 62 0 0 25 52M4150952 5.4 3 78 79 26 15 0 0 32 23

The T cell surface marker expression data in Table 10 provide acomparison of the immunomodulatory receptor profiles of tumorinfiltrating lymphocytes (TILS) from various human NSCLC tumor biopsysamples. Such data provided a useful biological context for the assaysperformed using the tumor samples.

C. Stimulation of Tumor Infiltrating Lymphocytes (TILs)

To establish polyclonal stimulation of TILs among the dissociated tumorcells, a 96-well assay plate was coated with 0.5 μg/mL anti-CD3 (OKT3)in coupling buffer, overnight at 4° C. The antibody coating solution wasremoved, and the plate was washed. Tumor suspensions were resuspended toa density of approximately 1.5×10⁶ cells per mL. Then 200 μL of this wasadded to each experimental well, together with 2 μg/mL anti-CD28 (clone28.2, eBioscience). At specific time points, supernatants were used forELISA analysis or cells were used for FACS analysis or single cellanalysis on microwell array devices.

D. Enzyme-Linked Immunosorbent Assay

Supernatants from cultured tumor digests containing tumor cells, stromalcells and immune cells were collected at fixed time points afterexperimental treatment, and cytokine production was assessed by ELISA.To begin the ELISA, 96-well plates were coated with capture antibody,blocked with assay diluent buffer, and washed, prior to incubation withserial dilutions of supernatants from the cultured tumor-derived cells.The samples were incubated for one hour, and then the ELISA plates werewashed. The detection antibody-HRP, in assay diluent, was then added,the assay plate was washed, and substrate solution was added to thewells in the assay plate. After the enzyme reaction was stopped,colorimetric density at 450 nm was measured in a conventional platereader. Measurements of IFNγ secretion, normalized to internal standardsincluded on each plate, was used as the experimental readout for T celleffector function.

E. TIL Function Increased by PD-1 Blockade

FIG. 12 summarizes results from an experiment showing restoration of Tcell function by PD-1 blockade with nivolumab or different humanizedforms of antibodies 388D4 and 244C8, i.e., 388D4-2, 388D4-3, 244C8-1,244C8-2, and 244C8-3. A population of 3×10⁵ cells, which included 17%lymphocytes (activated as described above) was incubated for 24 hourswith anti-PD-1 antibodies at a concentration of 20 μg/mL. IFNγ wasmeasured by ELISA, and the data were expressed in terms offold-activation relative to treatment with the isotype control antibody.As shown in FIG. 12, each of the anti-PD-1 antibodies restored T cellfunction, increasing IFNγ secretion approximately 7- to 8-fold, relativeto the isotype control. The data illustrated in FIG. 12 are presented inTable 11 below.

TABLE 11 Restoration of T cell function by PD-1 blockade Fold InductionStandard IFN-γ Deviation Isotype Control 1.00 0.39 nivolumab 7.07 0.15388D4-2 7.58 0.23 388D4-3 7.20 0.23 244C8-1 8.04 0.33 244C8-2 7.76 0.11244C8-3 7.79 0.42

FIG. 13 summarizes results from an experiment to measure the increase inT cell effector function, as indicated by IFNγ secretion, in response totreatment with antibody 244C8-2 alone versus treatment with 244C8-2 plus388D4-2, with results normalized relative to the response to treatmentwith nivolumab. A population of 3×10⁵ cells, which included 7.5%lymphocytes sub-optimally activated as described above, was incubatedfor 24 hours with anti-PD-1 antibodies at a total antibody concentrationof 20 μg/mL. As shown in FIG. 13, treatment with 244C8-2 alone increasedIFNγ 1.77-fold (±0.19 sd), while treatment with 244C8-2 in combinationwith 388D4-2 increased IFNγ secretion 2.11-fold (±0.21 sd). Theseunexpected results obtained in response to treatment with thecombination of a competitive inhibitory antibody and a non-competitiveinhibitory antibody indicate that combining these two differentmechanisms of action to inhibit PD-1 produces an enhanced response,despite the fact that both antibodies are directed against the sametarget. The data illustrated in FIG. 13 are presented in Table 12 below.

TABLE 12 Increase in IFN-γ induction by combination of anti-PD-1antibodies Fold Induction Standard IFN-γ Deviation Nivolumab 1.00 0.12244C8-2 1.77 0.19 388D4-2 + 244C8-2 2.11 0.21

FIG. 14 shows results from an experiment similar to the experimentyielding the results presented in FIG. 12, except that differenthumanized forms of antibodies 388D4 and 244C8 were tested, and nivolumabwas tested in combination with 244C8. In each treatment, a population of3×10⁵ cells, which included 9% lymphocytes (sub-optimally activated asdescribed above) was incubated for 24 hours with anti-PD-1 antibodies ata total concentration of 20 μg/mL. Following PD-1 blockade, cells andsupernatants were collected for FACS analysis, analysis bymicroengraving in a microwell array device (Varadarajan, supra), orELISA detection of cytokines. FIG. 14 shows IFNγ secretion, as measuredby ELISA, with data expressed in terms of fold-induction of IFNγsecretion, relative to treatment with the isotype control antibody. Thedata used to create FIG. 14 are shown in Table 13 below.

TABLE 13 Increase in IFN-γ induction by combination of anti-PD-1antibodies Fold Induction IFN-γ Standard Deviation anti-CD3 + anti-CD281.00 0.25 only Nivolumab (10 ug/mL) 1.89 0.07 388D4-2 (5 ug/mL) 2.060.12 388D4-2 (10 ug/mL) 1.82 0.38 244C8-2 (5 ug/mL) 2.66 0.49 244C8-2(10 ug/mL) 2.81 0.42 388D4-2 (5 ug/mL) + 2.85 0.55 244C8-2 (5 ug/mL)Nivolumab (5 ug/mL) + 3.76 0.08 244C8-2 (5 ug/mL)

The data summarized in FIG. 14 show significantly increased response totreatment of TILS from NSCLC biopsies when treated with the combinationof nivolumab, which is a competitive inhibitory anti-PD-1 antibody, and244C8-2, which is a non-competitive inhibitory anti-PD-1 antibody.Individual antibody treatment with each of two different anti-PD-1antibodies that compete with PD-L1 for binding to PD-L1, i.e., nivolumaband 388D4-2, increased T cell effector function approximately 2-fold.Individual antibody treatment with an anti-PD-1 antibody that does notcompete with PD-L1 for binding to PD-L1, i.e., 244C8-2, increased T celleffector function between 2.5-fold and 3-fold. Treatment with nivolumabplus 244C8-2 increased T cell effector function between 3.5-fold and4-fold.

When experiments such as these are performed on human patient tumorbiopsy samples, the magnitude of increase in T cell effector functionobserved in response to the same antibody treatment can vary fromexperiment to experiment as a function of patient-to-patient variation.In spite of such patient-to-patient variation, these results indicatethat the addition of a non-competitive inhibitory anti-PD-1 antibodytreatment to a competitive inhibitory anti-PD-1 antibody treatment canyield a greater increase in effector function of TILs, as compared totreatment with the competitive inhibitory anti-PD-1 antibody alone.

F. Antibody 244C8 and Cytokine Secretion in MLR Assays

Increased secretion of various cytokines was observed in response toantibody 244C8 in mixed lymphocyte reaction (MLR) assays. FIGS. 15A-15Fsummarize the results of an MLR assay performed on human PBMCs treatedwith anti-PD-1 antibodies. The MLR assay was performed usingcommercially available monocyte-derived dendritic cells as stimulatorcells and purified CD4+ T lymphocytes as responder cells from adifferent healthy blood donor. Supernatants were collected at 2.5 daysafter beginning the assay. Cytokine secretion was measured in amultiplex capture sandwich immunoassay using MagPlex® microspheres(Luminex, Austin, Tex.) and ProCartaPlex® Human TH1/TH2 Chemokine Panel(Luminex), according to the vendor's instructions. Fluorescence of thevarious labels was detected using a MagPix® fluorescence detectionsystem (Luminex). The data in FIGS. 15A-15F indicate that treatment withantibody 244C8-1 resulted in increased secretion of cytokines IL-6,IL-12, IL-18, TNF-α, GM-CSF, and IL-1β, in comparison with antibody388D4-2 or the IgG4 isotype control (Biolegend). Similar results (datanot shown) were observed with cells from two other blood donors, at twotime points.

G. Antibody 244C8 and Cytokine Secretion by TILs

Increased secretion of various cytokines by TILs was observed inresponse to antibody 244C8. FIGS. 16A-16F show alteration of tumorinfiltrating lymphocyte (TIL) function by PD-1 blockade with antibodies388D4-2, and 244C8-2. In this experiment, a population of 3×10⁵dissociated and suspended human cells from a non-small cell lung cancer(NSCLC) biopsy, which included 7% lymphocytes that had been activated asdescribed in Example 5C (above) was incubated for 24 hours with ananti-PD-1 antibody or IgG4 isotype control, at a concentration of 10μg/mL. Cytokine secretion was measured in a multiplex capture sandwichimmunoassay using MagPlex® microspheres (Luminex) and ProCartaPlex®Human TH1/TH2 Chemokine Panel (Luminex), according to the vendor'sinstructions. Fluorescence of the various labels was detected using aMagPix® fluorescence detection system (Luminex). These data indicatethat treatment with antibody 244C8-2 resulted in increased secretion ofcytokines IL-6, IL-12, IL-18, TNF-α, GM-CSF, and IL-1β, in comparisonwith antibody 388D4-2 or the IgG4 isotype control (Biolegend). Similarresults (not shown) were obtained by employing this protocol with threeother human tumor samples.

Example 6 Animal Models

A. Patient-Derived Xenografts in Humanized Mice

Antibodies 244C8 and 388D4 displayed anti-tumor activity inpatient-derived xenograft (PDX) tumor growth in humanized mice. FIG. 17Ashows results from an in vivo efficacy experiment involving a human lungtumor PDX-derived from a metastatic stage IV non-small cell lung cancer(NSCLC) patient (lung tumor LG1306; Jackson Labs) implanted in miceengineered to have a human immune system (hu-CD34 NSG™ mice; JacksonLabs). The five treatment groups were: vehicle control, antibody388D4-3, antibody 244C8-2, pembrolizumab, or a combination of antibody244C8-2 and pembrolizumab. Mice that had been engrafted with human CD34+cells and had >25% human CD45+ cells in the peripheral blood at twelveweeks post-engraftment were implanted subcutaneously on the right flankwith tumor fragments from PDX model LG1306. Mice were randomized intofive treatment groups (n=10), based on tumor volume, when volume reached60-120 mm³. Animals received a total of six intra-peritoneal 5 mg/kgdoses at five-day intervals (Q5D×6). All doses were delivered in PBS asvehicle. In the treatment groups that received 388D4-3, 244C8-2, orpembrolizumab, the first dose of the antibody was given as a 10 mg/kgdose, followed by the additional doses at the 5 mg/kg dose. Thecombination treatment group (244C8-2+pembrolizumab) received a dose eachof 5 mg/kg pembrolizumab and 5 mg/kg of 244C8-2 at each dosing timepoint. Tumor volumes were measured twice weekly (Day 3, 6, 10, 13, 17,20, 24 and 28) using a digital caliper to determine length and width ofthe tumors. Error bars represent the 95% confidence interval.

All treatment groups showed significant tumor growth inhibition comparedto the vehicle control group. In this experiment, no significantdifference in tumor growth inhibition was observed among treatment withantibody 388D4-3, antibody 244C8-2, pembrolizumab, or the combination ofantibody 244C8-2 with pembrolizumab. As shown in FIG. 17B, at day 28(end of study), the tumor volume for each of the treatment groups wassignificantly smaller than the vehicle group. The Student T-test pvalues between each treatment group and the vehicle group were:pembrolizumab=0.00167; 388D4=0.00105; 244C8=0.00277; and pembrolizumaband 244C8 in combination=0.00275. FIG. 17C shows percentage tumor volumeof each treatment group relative to vehicle on day 28. The calculatedpercent tumor growth inhibition (% TGI) for each treatment is shownabove each bar in FIG. 17C. Percent tumor growth inhibition wascalculated according to the following formula:

% TGI on Day X=(1−(T _(DayX) −T _(Day-1))/(C _(DayX) −C _(Day-1)))*100

where:T=the average tumor volume for a treatment group; andC=the average tumor volume for the control group.

In this experiment, it was surprisingly discovered that in thecombination treatment, a combined antibody dose totaling 10 mg/kg waswell tolerated by the animals, for the duration of the study.

INCORPORATION BY REFERENCE

The relevant teachings of all patents, published applications, andreferences cited herein are incorporated by reference in their entirety.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to examples of embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. An isolated antibody that binds to programmed cell death protein 1(PD-1), comprising a heavy chain variable region (HCVR) havingcomplementarity determining regions (CDRs) selected from the groupconsisting of: CDRs 1-3 of SEQ ID NO: 1; CDRs 1-3 of SEQ ID NO: 2; CDRs1-3 of SEQ ID NO: 3; CDRs 1-3 of SEQ ID NO: 4; CDRs 1-3 of SEQ ID NO: 5;CDRs 1-3 of SEQ ID NO: 6; CDRs 1-3 of SEQ ID NO: 7; CDRs 1-3 of SEQ IDNO: 8; CDRs 1-3 of SEQ ID NO: 9; CDRs 1-3 of SEQ ID NO: 10; CDRs 1-3 ofSEQ ID NO: 11; CDRs 1-3 of SEQ ID NO: 12; CDRs 1-3 of SEQ ID NO: 13;CDRs 1-3 of SEQ ID NO: 14; CDRs 1-3 of SEQ ID NO: 15; CDRs 1-3 of SEQ IDNO: 16; CDRs 1-3 of SEQ ID NO: 17; CDRs 1-3 of SEQ ID NO: 18; CDRs 1-3of SEQ ID NO: 19; CDRs 1-3 of SEQ ID NO: 20; CDRs 1-3 of SEQ ID NO: 21;CDRs 1-3 of SEQ ID NO: 22; CDRs 1-3 of SEQ ID NO: 23; CDRs 1-3 of SEQ IDNO: 24; CDRs 1-3 of SEQ ID NO: 25; and CDRs 1-3 of SEQ ID NO: 26, and alight chain variable region (LCVR) having CDRs selected from the groupconsisting of: CDRs 1-3 of SEQ ID NO: 27; CDRs 1-3 of SEQ ID NO: 28;CDRs 1-3 of SEQ ID NO: 29; CDRs 1-3 of SEQ ID NO: 30; CDRs 1-3 of SEQ IDNO: 31; CDRs 1-3 of SEQ ID NO: 32; CDRs 1-3 of SEQ ID NO: 33; CDRs 1-3of SEQ ID NO: 34; CDRs 1-3 of SEQ ID NO: 35; CDRs 1-3 of SEQ ID NO: 36;CDRs 1-3 of SEQ ID NO: 37; CDRs 1-3 of SEQ ID NO: 38; CDRs 1-3 of SEQ IDNO: 39; CDRs 1-3 of SEQ ID NO: 40; CDRs 1-3 of SEQ ID NO: 41; CDRs 1-3of SEQ ID NO: 42; CDRs 1-3 of SEQ ID NO: 43; CDRs 1-3 of SEQ ID NO: 44;CDRs 1-3 of SEQ ID NO: 45; CDRs 1-3 of SEQ ID NO: 46; CDRs 1-3 of SEQ IDNO: 47; CDRs 1-3 of SEQ ID NO: 48; CDRs 1-3 of SEQ ID NO: 49; CDRs 1-3of SEQ ID NO: 50; CDRs 1-3 of SEQ ID NO: 51; CDRs 1-3 of SEQ ID NO: 52;and CDRs 1-3 of SEQ ID NO:
 53. 2-5. (canceled)
 6. The antibody accordingto claim 1, wherein the antibody comprises a HCVR and LCVR pair selectedfrom the group consisting of: a HCVR having the sequence set forth inSEQ ID NO: 4 and a LCVR having the sequence set forth in SEQ ID NO: 28(244C7); a HCVR having the sequence set forth in SEQ ID NO: 4 and a LCVRhaving the sequence set forth in SEQ ID NO: 27 (244C7m1); a HCVR havingthe sequence set forth in SEQ ID NO: 1 and a LCVR having the sequenceset forth in SEQ ID NO: 28 (244C8); a HCVR having the sequence set forthin SEQ ID NO: 1 and a LCVR having the sequence set forth in SEQ ID NO:27 (244C8m1); a HCVR having the sequence set forth in SEQ ID NO: 3 and aLCVR having the sequence set forth in SEQ ID NO: 31 (246F7); a HCVRhaving the sequence set forth in SEQ ID NO: 5 and a LCVR having thesequence set forth in SEQ ID NO: 44 (258C1); a HCVR having the sequenceset forth in SEQ ID NO: 2 and a LCVR having the sequence set forth inSEQ ID NO: 30 (258F6); a HCVR having the sequence set forth in SEQ IDNO: 2 and a LCVR having the sequence set forth in SEQ ID NO: 29(258F6m); a HCVR having the sequence set forth in SEQ ID NO: 6 and aLCVR having the sequence set forth in SEQ ID NO: 34 (392C4); a HCVRhaving the sequence set forth in SEQ ID NO: 7 and a LCVR having thesequence set forth in SEQ ID NO: 41 (394D5); a HCVR having the sequenceset forth in SEQ ID NO: 8 and a LCVR having the sequence set forth inSEQ ID NO: 35 (394G1); a HCVR having the sequence set forth in SEQ IDNO: 12 and a LCVR having the sequence set forth in SEQ ID NO: 39(388C12A); a HCVR having the sequence set forth in SEQ ID NO: 12 and aLCVR having the sequence set forth in SEQ ID NO: 32 (388C12B); a HCVRhaving the sequence set forth in SEQ ID NO: 13 and a LCVR having thesequence set forth in SEQ ID NO: 39 (388C16A); a HCVR having thesequence set forth in SEQ ID NO: 13 and a LCVR having the sequence setforth in SEQ ID NO: 32 (388C16B); a HCVR having the sequence set forthin SEQ ID NO: 9 and a LCVR having the sequence set forth in SEQ ID NO:38 (392C5A); a HCVR having the sequence set forth in SEQ ID NO: 9 and aLCVR having the sequence set forth in SEQ ID NO: 37 (392C5B); a HCVRhaving the sequence set forth in SEQ ID NO: 17 and a LCVR having thesequence set forth in SEQ ID NO: 40 (392D2); a HCVR having the sequenceset forth in SEQ ID NO: 16 and a LCVR having the sequence set forth inSEQ ID NO: 43 (392H4); a HCVR having the sequence set forth in SEQ IDNO: 20 and a LCVR having the sequence set forth in SEQ ID NO: 53(246A10); a HCVR having the sequence set forth in SEQ ID NO: 18 and aLCVR having the sequence set forth in SEQ ID NO: 47 (388D4); a HCVRhaving the sequence set forth in SEQ ID NO: 19 and a LCVR having thesequence set forth in SEQ ID NO: 48 (392A6); a HCVR having the sequenceset forth in SEQ ID NO: 21 and a LCVR having the sequence set forth inSEQ ID NO: 52 (411C2); a HCVR having the sequence set forth in SEQ IDNO: 22 and a LCVR having the sequence set forth in SEQ ID NO: 51(413D2); and a HCVR having the sequence set forth in SEQ ID NO: 25 and aLCVR having the sequence set forth in SEQ ID NO: 45 (413E1). 7.(canceled)
 8. The antibody according to claim 1 comprising a humanizedor human framework region. 9-11. (canceled)
 12. The antibody accordingto claim 1, comprising a HCVR having the sequence set forth in SEQ IDNO: 90 and a LCVR having the sequence set forth in SEQ ID NO: 94; a HCVRhaving the sequence set forth in SEQ ID NO: 88 and a LCVR having thesequence set forth in SEQ ID NO: 96; a HCVR having the sequence setforth in SEQ ID NO: 90 and a LCVR having the sequence set forth in SEQID NO: 96; a HCVR having the sequence set forth in SEQ ID NO: 85 and aLCVR having the sequence set forth in SEQ ID NO: 91; a HCVR having thesequence set forth in SEQ ID NO: 85 and a LCVR having the sequence setforth in SEQ ID NO: 93; or a HCVR having the sequence set forth in SEQID NO: 86 and a LCVR having the sequence set forth in SEQ ID NO:
 91. 13.The antibody according to claim 1, wherein the antibody binds to asequence in PD-1 selected from the group consisting of amino acidresidues 74-104 and 115-139 of SEQ ID NO: 97; amino acid residues 74-139of SEQ ID NO: 97; and amino acid residues 4-63 and 94-123 of SEQ ID NO:97.
 14. (canceled)
 15. The antibody according to claim 1, wherein theantibody is a PD-1 agonist.
 16. The antibody according to claim 1,wherein the antibody is a PD-1 antagonist.
 17. An isolated antibody thatbinds to PD-1 and competitively inhibits the binding of the antibodyaccording to claim 6 selected from the group consisting of an antibodycomprising a HCVR having the sequence set forth in SEQ ID NO: 1 and aLCVR having the sequence set forth in SEQ ID NO: 28 (244C8); an antibodycomprising a HCVR having the sequence set forth in SEQ ID NO: 20 and aLCVR having the sequence set forth in SEQ ID NO: 53 (246A10); and anantibody comprising a HCVR having the sequence set forth in SEQ ID NO:22 and a LCVR having the sequence set forth in SEQ ID NO: 51 (413D2), tohuman PD-1.
 18. (canceled)
 19. (canceled)
 20. An isolated nucleic acidcomprising a nucleotide sequence encoding the HCVR of claim
 1. 21. Anexpression vector comprising the nucleic acid of claim
 20. 22. A hostcell transformed with an expression vector of claim
 21. 23-25.(canceled)
 26. A method of producing an antibody comprising a HCVR, aLCVR, or a combination thereof, the method comprising: (a) growing thehost cell of claim 22, under conditions such that the host cellexpresses the antibody comprising the HCVR, the LCVR, or a combinationthereof; and (b) isolating the antibody comprising the HCVR, the LCVR,or combination thereof.
 27. A method of treating a PD-1-mediated diseaseor disorder in a mammal, comprising administering an effective amount ofthe antibody according to claim 1 to a mammal in need thereof.
 28. Themethod of claim 27, wherein the mammal has an immune disorder or cancer.29. A method of diagnosing a PD-1-mediated adaptive immune resistance ina patient who has cancer, comprising: contacting a tumormicroenvironment in the patient with the antibody according to claim 1labeled with a detectable moiety; and detecting expression of PD-1 onCD8+ T cells within the tumor microenvironment.
 30. (canceled)
 31. Amethod for increasing T cell effector function, comprising contacting aT cell with a combination of: (a) an effective amount of an anti-PD-1antibody that competitively inhibits binding of PD-L1 or PD-L2 to PD-1expressed on the surface of the T cell; and (b) an effective amount ofan anti-PD-1 antibody that does not competitively inhibit binding ofPD-L1 or PD-L2 to PD-1 expressed on the surface of the T cell. 32.(canceled)
 33. (canceled)
 34. A method of treating cancer in a mammal,comprising contacting a T cell in a mammal in need thereof with acombination of: (a) an effective amount of an anti-PD-1 antibody thatcompetitively inhibits binding of PD-L1 or PD-L2 to PD-1 expressed onthe surface of the T cell; and (b) an effective amount of an anti-PD-1antibody that does not competitively inhibit binding of PD-L1 or PD-L2to PD-1 expressed on the surface of the T cell.
 35. The method of claim34, wherein the cancer is selected from the group consisting of:melanoma, renal cancer, prostate cancer, pancreatic adenocarcinoma,breast cancer, colon cancer, lung cancer, esophageal cancer, squamouscell carcinoma of the head and neck, liver cancer, ovarian cancer,cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, andlymphoma.
 36. (canceled)
 37. The method of claim 31, wherein theanti-PD-1 antibody that competitively inhibits binding of PD-L1 or PD-L2to PD-1 expressed on the surface of a T cell is selected from the groupconsisting of antibody 388D4-1, antibody 388D4-2 and antibody 388D4-3.38. The method of claim 1, wherein the anti-PD-1 antibody that does notcompetitively inhibit binding of PD-L1 or PD-L2 to PD-1 expressed on thesurface of a T cell is selected from the group consisting of antibody244C8-1, antibody 244C8-2 and antibody 244C8-3.
 39. (canceled) 40.(canceled)
 41. The method of claim 31, wherein the anti-PD-1 antibodythat does not competitively inhibit binding of PD-L1 or PD-L2 to PD-1expressed on the surface of the T cell binds to a PD-1 epitope bound by244C8.
 42. The method of claim 31, wherein the anti-PD-1 antibody thatdoes not competitively inhibit binding of PD-L1 or PD-L2 to PD-1expressed on the surface of the T cell competes with 244C8 for bindingto PD-1.
 43. A method for increasing T cell effector function,comprising contacting a T cell with an anti-PD-1 antibody that does notcompetitively inhibit binding of PD-L1 or PD-L2 to PD-1 expressed on thesurface of the T cell.
 44. A method for increasing lymphocyte secretionof a cytokine selected from the group consisting of IL-6, IL-12, IL-18,TNF-α, IL-1β and GM-CSF in a human patient in need of increased T celleffector function, comprising administering to the patient atherapeutically effective amount of an anti-PD-1 antibody that does notcompetitively inhibit binding of PD-L1 or PD-L2 to PD-1 expressed on thesurface of a T cell.
 45. (canceled)
 46. (canceled)
 47. The method ofclaim 44, wherein the antibody is selected from the group consisting ofantibody 244C8-1, antibody 244C8-2 and antibody 244C8-3.
 48. Theantibody according to claim 1, comprising a HCVR having CDRs 1-3 of SEQID NO: 1, and a LCVR having CDRs 1-3 of SEQ ID NO:
 28. 49. The antibodyaccording to claim 1, comprising a HCVR having CDRs 1-3 of SEQ ID NO:18, and a LCVR having CDRs 1-3 of SEQ ID NO:
 47. 50. The method of claim37, wherein the anti-PD-1 antibody that competitively inhibits bindingof PD-L1 or PD-L2 to PD-1 expressed on the surface of a T cell isselected from the group consisting of antibody 388D4-1, antibody 388D4-2and antibody 388D4-3.
 51. The method of claim 34, wherein the anti-PD-1antibody that competitively inhibits binding of PD-L1 or PD-L2 to PD-1expressed on the surface of a T cell is selected from the groupconsisting of antibody 388D4-1, antibody 388D4-2 and antibody 388D4-3.52. The method of claim 34, wherein the anti-PD-1 antibody that does notcompetitively inhibit binding of PD-L1 or PD-L2 to PD-1 expressed on thesurface of a T cell is selected from the group consisting of antibody244C8-1, antibody 244C8-2 and antibody 244C8-3.
 53. The method of claim51, wherein the anti-PD-1 antibody that competitively inhibits bindingof PD-L1 or PD-L2 to PD-1 expressed on the surface of a T cell isselected from the group consisting of antibody 388D4-1, antibody 388D4-2and antibody 388D4-3.